Implemented Microscopes
As an example of the flexibility of the navigate software, we describe several microscope implementations that are currently in use with the software. Each of these implementations is described in the following sections, including a list of the equipment used and the configuration file used to operate the microscope. While this list does not aim to be comprehensive, it does show the diversity of microscopes that can be controlled using the navigate software. For example, this includes single and multi-camera systems, microscopes with different illumination and detection schemes, and microscopes with different sample geometries and scan mechanisms.
For more information on how to build these microscopes, as well as their performance, please refer to the publications listed in the relevant sections.
ASLM
(Top) Collagen-embedded cells imaged with ASLM. Segmented cells shown on the left, and the corresponding raw data shown on the right. Data from Driscoll et al, Nat. Methods. 2019. Scale bars, 10 microns. (Bottom) images of the live-cell ASLM implementation.
Axially Swept Light-Sheet Microscope (ASLM) is designed specifically to achieve high-resolution imaging throughout large fields of view. This custom-built system offers unparalleled resolution of 300-380 nm isotropically across extensive imaging volumes measuring up to 200x200x200 microns, employing one-photon excitation techniques.
The ASLM system features a Special Optics 28.5X/NA 0.67 illumination objective and a Nikon 25X/NA 1.1 detection objective. It is equipped with a diverse array of laser wavelengths including 445, 488, 514, 561, and 642 nm, enabling versatile multicolor imaging capabilities. The system leverages aberration-free remote focusing to scan the illumination beam in sync with the rolling shutters of two Hamamatsu Flash 4.0 sCMOS cameras. Because ASLM does not depend upon diffractive optics, such as spatial light modulators, it is capable of simultaneous multicolor imaging.
Unique to ASLM, the system maintains diffraction-limited and isotropic resolution over arbitrarily large volumes. The resulting point spread function (PSF) is isotropic, devoid of side lobes or significant blurring, obviating the need for deconvolution. Moreover, ASLM achieves significantly higher illumination uniformity compared to traditional Gaussian or Bessel beam approaches. This uniformity translates to a consistent signal-to-noise ratio (SNR) throughout the imaging volume, greatly enhancing quantitative imaging applications such as fluorescence resonance energy transfer and single particle tracking. This combination of features makes ASLM an exceptional tool for detailed, quantitative imaging across large sample volumes.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Deconvolution-free subcellular imaging with axially swept light sheet microscopy
Diagonally scanned light-sheet microscopy for fast volumetric imaging of adherent cells
u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions
Robust and automated detection of subcellular morphological motifs in 3D microscopy images
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Coherent Obis with emission at 405, 457, 488, 514, 561, and 642 nm. |
Stages |
MP-285, PI P-726 PIFOC High-Load piezo, and a galvo for acquisition of z-stacks. |
Stage Controllers |
Sutter MP-285 and PI E-709 |
Cameras |
2x Hamamatsu Flash 4.0 |
Filter Wheel |
Sutter Lambda 10-3 with 1x 25mm Filter Wheels |
Remote Focusing Units |
Equipment Solutions LFA-2010 Linear Focus Actuator |
Data Acquisition Cards |
National Instruments PCIe-6738 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo |
Zoom |
N/A |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
CTASLMv2:
daq:
hardware:
type: NI
sample_rate: 100000
# triggers
master_trigger_out_line: PCI6738/port0/line1 #3
camera_trigger_out_line: /PCI6738/ctr0 #9/42
trigger_source: /PCI6738/PFI0 #4
camera:
# -
hardware:
type: HamamatsuOrca
serial_number: 100803
defect_correct_mode: 2.0
delay: 20 #ms
settle_down: 0.0 #ms
# -
# hardware:
# name: camera
# type: HamamatsuOrca
# serial_number: 003054
# x_pixels: 2048.0
# y_pixels: 2048.0
# pixel_size_in_microns: 6.5
# subsampling: [1, 2, 4]
# sensor_mode: Normal # 12 for progressive, 1 for normal. Normal/Light-Sheet
# readout_direction: Top-to-Bottom # Top-to-Bottom', 'Bottom-to-Top'
# lightsheet_rolling_shutter_width: 608
# defect_correct_mode: 2.0
# binning: 1x1
# readout_speed: 2.0
# trigger_active: 1.0
# trigger_mode: 1.0 # external light-sheet mode
# trigger_polarity: 2.0 # positive pulse
# trigger_source: 2.0 # 2 = external, 3 = software.
# exposure_time: 20 # Use milliseconds throughout.
# delay_percent: 20
# pulse_percent: 1
# line_interval: 0.000075
# display_acquisition_subsampling: 4
# display_acquisition_subsampling: 4
# average_frame_rate: 4.969
# frames_to_average: 1
# exposure_time_range:
# min: 1
# max: 1000
# step: 1
remote_focus_device:
hardware:
type: NI #synthetic #EquipmentSolutions # NI
channel: PCI6738/ao2 #45/46
port: COM700
min: -5
max: 5
galvo:
-
hardware:
type: NI
channel: PCI6738/ao0 #10/11
min: -5
max: 5
waveform: sawtooth
phase: 1.57079 # pi/2
filter_wheel:
hardware:
type: SutterFilterWheel
wheel_number: 1
port: COM1
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
647-LP: 0
600-50: 1
525-50: 2
480-40: 3
Empty-1: 4
Empty-2: 5
Empty-3: 6
Empty-4: 7
stage:
hardware:
-
name: stage1
type: synthetic #MP285
serial_number: 0001
port: COM6
timeout: 0.25
baudrate: 9600
axes: [y, x, f]
axes_mapping: [z, y, x]
max: 25000
min: 0
-
name: stage2
type: syntheticstage
serial_number: 0000
axes: [theta]
axes_mapping: [theta]
max: 360
min: 0
-
name: PIFOC
type: GalvoNIStage
serial_number: 0000
axes: [z]
axes_mapping: [PCI6738/ao6] #48/49
volts_per_micron: 0.1*x
max: 10
min: 0
x_max: 12500
x_min: -12500
y_max: 12500
y_min: -12500
z_max: 200
z_min: 0
f_max: 12500
f_min: -12500
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
theta_offset: 0
f_offset: 0
zoom:
hardware:
type: synthetic
servo_id: 1
port: COM18
baudrate: 10000
position:
62.5X: 0.104
pixel_size:
62.5X: 0.104
stage_positions:
BABB:
f:
62.5X: 0
shutter:
hardware:
type: SyntheticShutter
channel: PCI6738/port0/line10
min: 0
max: 5
lasers:
- wavelength: 405
onoff:
hardware:
type: NI
channel: PCI6738/port1/line5 # 7/41
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao8 # 16/50
min: 0
max: 5
type: Obis
- wavelength: 445
onoff:
hardware:
type: NI
channel: PCI6738/port1/line2 # 5/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao9 # 17/50
min: 0
max: 5
type: Obis
- wavelength: 488
onoff:
hardware:
type: NI
channel: PCI6738/port1/line3 # 6/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao10 # 51/50
min: 0
max: 5
type: Obis
- wavelength: 514
onoff:
hardware:
type: NI
channel: PCI6738/port1/line4 #40/41
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao11 #52/18
min: 0
max: 5
type: Obis
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCI6738/port1/line6 # 8/42
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao12 #53/20
min: 0
max: 5
type: Obis
- wavelength: 640
onoff:
hardware:
type: NI
channel: PCI6738/port0/line0 #9/42
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao13 #54/20
min: 0
max: 5
type: Obis
gui:
channels:
count: 5
Optical Tiling OPM
(Left) Image of a monolayer of keratinoctyes imaged with the Optical Tiling OPM. Region shown with a red box is magnified and shown through time below. (Right, top and bottom) Images of the Optical Tiling OPM system.
The Oblique Plane Microscope (OPM) described here is engineered for high-resolution, fast volumetric imaging of large fluorescent samples, such as cell monolayers, spheroids, and zebrafish embryos. This system addresses the common challenge of achieving high spatiotemporal resolution throughout a large field of view, which is often restricted in OPM setups due to optics associated with the tertiary imaging system.
To overcome these constraints, the microscope incorporates a novel dual-axis scan unit, enabling rapid, high-resolution imaging across a volume of 800 × 500 × 200 microns. Furthermore, the system integrates a multi-perspective projection imaging technique, which significantly enhances the volumetric interrogation rate to over 10 Hz. This feature facilitates quick scanning across a large field of view in a dimensionally reduced format, allowing for the swift identification and detailed imaging of specific regions of interest.
The custom-built oblique plane microscope is built in an inverted geometry. Illumination is provided obliquely at a 40-degree angle using an Olympus 20X/NA 1.05 water immersion objective, and fluorescence is captured in an epi-fluorescence format by the same objective. The fluorescence signal is then relayed through an Olympus 20x/NA 0.8 secondary objective and redirected by a custom glass-tipped tertiary objective set at a 40-degree angle. Images are captured using a Hamamatsu Flash 4.0 sCMOS camera. Multi-well plate plate imaging is available through mechanical sample scanning.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Coherent Galaxy with 488, 561, and 642 nm lasers. |
Stages |
ASI FTP-2000 with MS-2000 XY stage, and a Galvo for acquisition of z-stacks. |
Stage Controllers |
ASI Tiger Controller |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
2x ASI 6-Position 32 mm Filter Wheels |
Remote Focusing Units |
Optotune Electrotunable Lens (EL-16-40-TC-VIS-5D-1-C) |
Data Acquisition Cards |
National Instruments PCIe-6738 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo, and 2x Novanta Linear Galvos for shearing and tiling. |
Zoom |
N/A |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
OPMv2:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: /Dev5/port0/line1
camera_trigger_out_line: /Dev5/ctr0
trigger_source: /Dev5/PFI0
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
1x: 0
pixel_size:
1x: 0.15
shutter:
hardware:
type: SyntheticShutter
channel: none/line0
camera:
hardware:
name: camera
type: HamamatsuOrca
serial_number: 304064
defect_correct_mode: 2.0
delay: 7.5 #ms
settle_down: 0.0 #ms
remote_focus_device:
hardware:
type: NI
channel: Dev5/ao3
min: 0
max: 5
# Optotune EL-16-40-TC-VIS-5D-1-C
galvo:
# -
# name: xgalvo
# hardware:
# name: daq
# type: NI
# channel: Dev5/ao0
# min: -5
# max: 5
# frequency: 99.9
# amplitude: 2.5
# offset: 0
# duty_cycle: 50
# phase: 1.57079 # pi/2
-
name: ygalvo
hardware:
type: NI
channel: Dev5/ao1
min: -5
max: 5
phase: 1.57079 # pi/2
-
name: sheargalvo
hardware:
name: daq
type: NI
channel: Dev5/ao2
min: -5
max: 5
phase: 1.57079 # pi/2
stage:
hardware:
-
name: fake_stage
type: SyntheticStage
serial_number: 123
axes: [x,y,theta,f]
-
name: galvo-stage
type: GalvoNIStage
serial_number: 123
axes: [z]
axes_mapping: [Dev5/ao0]
min: -5
max: 5
volts_per_micron: 0.01*x + 0
x_max: 500
x_min: -500
y_max: 500
y_min: -500
z_max: 500
z_min: -500
f_max: 100000
f_min: -100000
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
filter_wheel:
hardware:
type: SyntheticFilterWheel
wheel_number: 1
port: COM6
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
FRFP - BLP01-664R-25: 0
RFP - FF01-598/25-25: 1
GFP - 527/20: 2
GFPRFP - ZET488/561m: 3
Empty-Alignment: 4
Blocked2: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 488
power:
hardware:
type: NI
channel: Dev5/ao12
min: 0
max: 5
type: LuxX
- wavelength: 562
onoff:
hardware:
type: NI
channel: Dev5/port1/line5
min: 0
max: 5
power:
hardware:
type: NI
channel: Dev5/ao13
min: 0
max: 5
type: Obis
- wavelength: 642
power:
hardware:
type: NI
channel: Dev5/ao14
min: 0
max: 5
type: LuxX
gui:
channels:
count: 5
AO OPM
(Left) Images of zebrafish vasculature before and after application of sensorless adaptive optics. (Right) image of the OPM equipped with adaptive optics.
Adaptive optics (AO) significantly enhances the performance of microscopes by restoring diffraction-limited imaging capabilities, particularly beneficial in light-sheet fluorescence microscopy (LSFM) where optical aberrations can vary significantly between the illumination and detection paths. To overcome these challenges, this oblique plane microscope includes a singular deformable mirror that effectively corrects aberrations in both the illumination and detection paths simultaneously. Aberrations are measured in a sensorless format on projection images, which stabilizes and refines wavefront corrections.
The custom-built OPM is optimized for zebrafish studies and incorporates a VAST BioImager Platform that automates zebrafish loading and positioning. Illumination is provided obliquely at 40 degrees using a Nikon 25X/NA 1.1 water immersion objective, and the collected fluorescence is processed using the same objective in an epi-fluorescence format. The signal is then relayed through an Olympus 20x/NA 0.8 secondary objective, directed by a custom glass-tipped tertiary objective at a 40-degree angle, and finally captured on a Hamamatsu Flash 4.0 sCMOS camera.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Omicron LightHUB Ultra with 488 and 561 nm lasers. |
Stages |
A piezo for adjusting the position of the tertiary objective, and a galvo for acquisition of z-stacks. |
Stage Controllers |
N/A |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
N/A |
Remote Focusing Units |
N/A |
Data Acquisition Cards |
National Instruments PCIe-6738 |
Galvo |
Two Novanta galvos for shearing and lateral sweeping of the illumination beam. |
Zoom |
N/A |
Other |
VAST large object flow cytometry system and Imagine Optics deformable mirror for wavefront correction. |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
ProjectionScope:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: /PCIe-6738/port0/line1
camera_trigger_out_line: /PCIe-6738/ctr0 #PFI7 Camera trigger
trigger_source: /PCIe-6738/PFI0
mirror:
hardware:
name: mirror
type: ImagineOpticsMirror
flat_path: D:\WaveKitX64\MirrorFiles\BeadsCoverslip_20231212.wcs
n_modes: 32
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
1x: 0
pixel_size:
1x: 0.15
shutter:
hardware:
type: SyntheticShutter
channel: none/line0
camera:
hardware:
name: camera
type: HamamatsuOrca
serial_number: 001301
defect_correct_mode: 2.0
delay: 10 #ms
settle_down: 0.0 #ms
remote_focus_device:
hardware:
type: SyntheticRemoteFocus
channel: none
min: 0
max: 5
galvo:
-
# shear galvo measured: 351.04 um/V
name: sheargalvo
hardware:
type: NI # SyntheticGalvo
channel: PCIe-6738/ao12
min: -5
max: 5
# waveform: halfsaw
waveform: sawtooth
phase: 1.57079
-
name: xgalvo
hardware:
type: NI # SyntheticGalvo
channel: PCIe-6738/ao0
min: -5
max: 5
waveform: sawtooth
phase: 1.57079
stage:
hardware:
-
name: fake_stage
type: SyntheticStage
serial_number: 123
axes: [x,y,theta,z]
-
name: snouty_piezo
type: GalvoNIStage
serial_number: 124
axes: [f]
axes_mapping: [PCIe-6738/ao14]
min: 0
max: 10
volts_per_micron: (10/15.4)*x + 5.0
x_max: 50
x_min: -50
y_max: 50
y_min: -50
z_max: 500
z_min: -500
f_max: 100000
f_min: -100000
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
filter_wheel:
hardware:
type: SyntheticFilterWheel
wheel_number: 1
port: COM6
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
FRFP - BLP01-664R-25: 0
RFP - FF01-598/25-25: 1
GFP - 527/20: 2
GFPRFP - ZET488/561m: 3
Empty-Alignment: 4
Blocked2: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 488
onoff:
hardware:
type: NI
channel: PCIe-6738/port1/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PCIe-6738/ao5
min: 0
max: 5
type: Obis
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCIe-6738/port1/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PCIe-6738/ao11
min: 0
max: 5
type: Obis
StackingScope:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: /PCIe-6738/port0/line1
camera_trigger_out_line: /PCIe-6738/ctr0 #PFI7 Camera trigger
trigger_source: /PCIe-6738/PFI0
mirror:
hardware:
name: mirror
type: ImagineOpticsMirror
flat_path: D:\WaveKitX64\MirrorFiles\BeadsCoverslip_20231212.wcs
n_modes: 32
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
1x: 0
pixel_size:
1x: 0.15
shutter:
hardware:
name: shutter
type: SyntheticShutter
channel: none/line0
camera:
hardware:
name: camera
type: HamamatsuOrca
serial_number: 001301
defect_correct_mode: 2.0
delay: 10 #ms
remote_focus_device:
hardware:
type: SyntheticRemoteFocus
channel: none
min: 0
max: 10
# Optotune EL-16-40-TC-VIS-5D-1-C
galvo:
-
name: sheargalvo
hardware:
type: NI # SyntheticGalvo
channel: PCIe-6738/ao12
min: -5
max: 5
phase: 1.57079
stage:
hardware:
-
name: fake_stage
type: SyntheticStage
serial_number: 123
axes: [x,y,theta]
-
name: snouty_piezo
type: GalvoNIStage
serial_number: 124
axes: [f]
axes_mapping: [PCIe-6738/ao14]
min: 0
max: 10
volts_per_micron: (10/15.4)*x + 5.0
-
name: z_galvo
type: GalvoNIStage
serial_number: 125
axes: [z]
# axes_channels: [PCIe-6738/ao0]
axes_mapping: [PCIe-6738/ao0]
min: -3.5
max: 3.5
volts_per_micron: 0.007*x
x_max: 50
x_min: -50
y_max: 50
y_min: -50
z_max: 500
z_min: -500
f_max: 100000
f_min: -100000
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
filter_wheel:
hardware:
type: SyntheticFilterWheel
wheel_number: 1
port: COM6
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
FRFP - BLP01-664R-25: 0
RFP - FF01-598/25-25: 1
GFP - 527/20: 2
GFPRFP - ZET488/561m: 3
Empty-Alignment: 4
Blocked2: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 488
onoff:
hardware:
type: NI
channel: PCIe-6738/port1/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PCIe-6738/ao5
min: 0
max: 5
type: Obis
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCIe-6738/port1/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PCIe-6738/ao11
min: 0
max: 5
type: Obis
gui:
channels:
count: 5
CT ASLM - v1
Image of a CLARITY-cleared mouse hippocampus imaged with the CT ASLM. Specimen was placed in glycerol, which results in slight swelling of the specimen, thereby improving the imaging resolution.
The Cleared Tissue Axially Swept Light-Sheet Microscope (CT ASLM) - Version 1 combines subcellular detail with tissue-scale anatomical views. This microscope provides an isotropic resolution of ~700 nm, which is provides insight into complex 3D morphologies in large tissue contexts. It achieves a field of view of 870 x 870 microns in water and 737 x 737 microns in BABB, and is equipped with NA 0.4 multi-immersion objectives from Applied Scientific Instrumentation. Illumination is provided with Coherent OBIS solid-state lasers with wavelengths of 405 nm, 488 nm, 561 nm, and 637 nm. Images are acquired with a Hamamatsu ORCA Flash 4.0 sCMOS camera. It is also equipped with a fast filter wheel and 3D motorized stage from Sutter Instruments, which enables tiling of large volumes. For acquisition of Z-stacks, the microscope uses a piezo stage from Mad City Labs.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Isotropic imaging across spatial scales with axially swept light-sheet microscopy
Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Coherent Obis lasers with emission at 488, 561, and 642 nm. |
Stages |
MP-285 and Piezo Jena 200-micron piezo for acquisition of z-stacks via sample scanning. |
Stage Controllers |
Sutter MP-285 |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
Sutter Lambda 10-3 with 1x 25mm Filter Wheel |
Remote Focusing Units |
Equipment Solutions LFA-2010 Linear Focus Actuator |
Data Acquisition Cards |
National Instruments PCIe-6738 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo |
Zoom |
N/A |
Other |
NA |
Configuration File
microscopes:
CTASLMv1:
daq:
hardware:
type: NI
sample_rate: 100000
# triggers
master_trigger_out_line: PCI6738/port0/line1
camera_trigger_out_line: /PCI6738/ctr0
trigger_source: /PCI6738/PFI0
# Digital Laser Outputs
laser_port_switcher: PCI6738/port0/line0
laser_switch_state: False
camera:
hardware:
type: HamamatsuOrca
serial_number: 000420
defect_correct_mode: 2.0
delay: 2.0 #ms
settle_down: 0.0 #ms
flip_x: False
flip_y: False
remote_focus_device:
hardware:
type: EquipmentSolutions # NI
channel: PCI6738/ao2 #45/46
port: COM1
baudrate: 9600
min: -5
max: 5
galvo:
-
hardware:
type: NI
channel: PCI6738/ao0 #10/11
min: -5
max: 5
waveform: sawtooth
phase: 1.57079 # pi/2
filter_wheel:
hardware:
type: SutterFilterWheel
wheel_number: 1
port: COM34
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
445-20: 6
525-30: 0
550-49: 9 # switched
600-53: 7
665LP: 8
EMPTY: 1
BLOCKED1: 2
BLOCKED2: 3
BLOCKED3: 4
BLOCKED4: 5
# 665LP: 0
# 550-49: 1
# 525-30: 2
# 445-20: 3
# Blocked1: 4
# Blocked2: 5
# Blocked3: 6
# Blocked4: 7
# Blocked5: 8
# Blocked6: 9
stage:
hardware:
-
type: MP285
serial_number: 0000
axes: [y, x, f]
axes_mapping: [z, y, x]
volts_per_micron: None
max: 25000
min: 0
port: COM2
timeout: 0.25
baudrate: 9600
-
type: syntheticstage
serial_number: 0000
axes: [theta]
axes_mapping: [theta]
max: 360
min: 0
-
type: GalvoNIStage
serial_number: 0000
axes: [z]
axes_mapping: [PCI6738/ao6]
volts_per_micron: 0.02*x
max: 10
min: 0
# joystick_axes: [x, y, f]
x_max: 12500
x_min: -12500
y_max: 12500
y_min: -12500
z_max: 500
z_min: 0
f_max: 100000
f_min: -100000
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
theta_offset: 0
f_offset: 0
zoom:
hardware:
type: synthetic
servo_id: 1
position:
16X: 0
pixel_size:
16X: 0.425
stage_positions:
BABB:
f:
16X: 0
shutter:
hardware:
type: synthetic
channel: PCI6738/port0/line10
min: 0
max: 5
lasers:
- wavelength: 642
onoff:
hardware:
type: NI
channel: PCI6738/port1/line5 # 38/35
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao8 #16/50 #ao1 # 44/11
min: 0
max: 5
type: Obis
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCI6738/port1/line2 # 5/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao9 #17/50 #ao3 # 12/46
min: 0
max: 5
type: Obis
- wavelength: 488
onoff:
hardware:
type: NI
channel: PCI6738/port1/line3 # 6/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao10 #51/50 #ao4 13/14
min: 0
max: 5
type: Obis
- wavelength: 405
onoff:
hardware:
type: NI
channel: PCI6738/port1/line4 # 40/41
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao11 #52/18 #ao5 #47/14
min: 0
max: 5
type: Obis
gui:
channels:
count: 5
CT ASLM - v2
Image of the peripheral nervous system in the hematopoietic stem cell niche. Nerves are shown in green, and hematopoietic progenitor cells in magenta.
This microscope is engineered to achieve an isotropic resolution of 300 nm throughout a field of view of ~340 x 340 microns. It includes high-performance Coherent OBIS solid-state lasers at wavelengths of 488 nm, 561 nm, and 637 nm. Imaging is performed with a Hamamatsu ORCA Flash 4.0 sCMOS camera and a fast filter wheel from Sutter Instruments. The microscope is equipped with 2x NA 0.7 multi-immersion objectives from Applied Scientific Instrumentation. Sample positioning is handled by a Sutter Instruments 3D motorized stage, which supports the tiling of large volumes. Z-stacks are acquired with a 200-micron Piezosystem Jena stage.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Isotropic imaging across spatial scales with axially swept light-sheet microscopy
Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Coherent Obis lasers with emission at 405, 488, 561, and 642 nm. |
Stages |
Sutter MP-285 and Mad City Lab 500-micron piezo for acquisition of z-stacks via sample scanning. |
Stage Controllers |
Sutter MP-285 |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
Sutter Lambda 10-3 with 1x 25mm Filter Wheel |
Remote Focusing Units |
Equipment Solutions LFA-2010 Linear Focus Actuator |
Data Acquisition Cards |
National Instruments PCIe-6738 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo |
Zoom |
N/A |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
CTASLMv2:
daq:
hardware:
type: NI
sample_rate: 100000
# triggers
master_trigger_out_line: PCI6738/port0/line1 #3
camera_trigger_out_line: /PCI6738/ctr0 #9/42
trigger_source: /PCI6738/PFI0 #4
# Digital Laser Outputs
laser_port_switcher: PCI6738/port0/line0
laser_switch_state: False
camera:
hardware:
type: HamamatsuOrca
serial_number: 302153
flip_x: True
flip_y: False
defect_correct_mode: 2.0
delay: 20
remote_focus_device:
hardware:
type: EquipmentSolutions # NI
channel: PCI6738/ao2 #45/46
port: COM7
min: -5
max: 5
galvo:
-
hardware:
type: NI
channel: PCI6738/ao0 #10/11
min: -5
max: 5
waveform: sawtooth
phase: 1.57079 # pi/2
filter_wheel:
hardware:
type: SutterFilterWheel
wheel_number: 1
port: COM10
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
Empty-1: 0
525-30: 1
600-52: 2
670-30: 3
647-LP: 4
Empty-2: 5
Empty-3: 6
Empty-4: 7
stage:
hardware:
-
name: stage1
type: MP285
serial_number: 0000
axes: [y, x, f]
axes_mapping: [z, y, x]
volts_per_micron: None
max: 25000
min: 0
port: COM2
timeout: 0.25
baudrate: 9600
-
name: stage2
type: syntheticstage
serial_number: 0000
axes: [theta]
axes_mapping: [theta]
max: 360
min: 0
-
name: stage3
type: GalvoNIStage
serial_number: 0000
axes: [z]
axes_mapping: [PCI6738/ao6] #48/49
volts_per_micron: 0.05*x
max: 10
min: 0
distance_threshold: 20
settle_duration_ms: 100
x_max: 12500
x_min: -12500
y_max: 12500
y_min: -12500
z_max: 200
z_min: 0
f_max: 12500
f_min: -12500
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
theta_offset: 0
f_offset: 0
coupled_axes:
z: f
zoom:
hardware:
type: synthetic
servo_id: 1
position:
36X: 0
pixel_size:
36X: 0.180
stage_positions:
BABB:
f:
36X: 0
shutter:
hardware:
type: SyntheticShutter
channel: PCI6738/port0/line10
min: 0
max: 5
lasers:
- wavelength: 488
onoff:
hardware:
type: NI
channel: PCI6738/port1/line5 # 7/41
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao8 #1 # 44/11
min: 0
max: 5
type: Obis
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCI6738/port1/line2 # 5/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao9 # 3 # 12/46
min: 0
max: 5
type: Obis
- wavelength: 647
onoff:
hardware:
type: NI
channel: PCI6738/port1/line3 # 6/39
min: 0
max: 5
power:
hardware:
type: NI
channel: PCI6738/ao10 # 4 # 13/14
min: 0
max: 5
type: Obis
gui:
channels:
count: 5
Expansion ASLM
(Left) Image of an expanded liver section imaged with the Expansion ASLM. (Right) Image of the Expansion ASLM system.
This upright variant of Axially Swept Light-Sheet Microscopy (ASLM) boasts a field of view that is 3.2 times larger than its predecessors, measuring 774 x 435 microns with a raw and isotropic resolution of approximately 420 nm. The upright sample geometry is advantageous for imaging fragile, expanded tissues, as well as samples that have large lateral extents. The system is equipped with an Omicron LightHub Ultra laser launch that features multiple fiber outputs and provides emission wavelengths at 405, 488, 561, and 642 nm. The microscope utilizes a high-sensitivity back-thinned Hamamatsu Lightning sCMOS camera and includes a fast filter wheel. Imaging is performed with 0.7 NA multi-immersion objectives and a FTP-2000 motorized stage from Applied Scientific Instrumentation. The microscope supports multiple imaging modes to accommodate different research needs. These include the classical step-and-settle routine, where the stage moves between image acquisitions. For large samples, the stage can operate at a constant velocity, with images captured at predetermined intervals, minimizing the latency typically associated with the step-and-settle method and improving throughput. Lastly, the microscope can operate in a mechanically sheared acquisition format, where two stages are scanned simultaneously. This approach aligns data automatically in its correct spatial context, significantly reducing the need for computational post-processing and eliminating data interpolation and duplication.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.
Isotropic imaging across spatial scales with axially swept light-sheet microscopy
Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Omicron LightHUB Ultra with 405, 488, 561, and 642 nm lasers. |
Stages |
ASI FTP-2000 with Linear Encoders in X and Y, and 3x LS-50 Linear Stages |
Stage Controllers |
ASI Tiger Controller |
Cameras |
Hamamatsu Lightning and Photometrics Iris15 |
Filter Wheel |
2x ASI 6-Position 32 mm Filter Wheels |
Remote Focusing Units |
ThorLabs BLINK |
Data Acquisition Cards |
National Instruments PXIe-1073 chassis equipped with PXI6733 and PXI6259 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo |
Zoom |
N/A |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
Nanoscale:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: PXI6259/port0/line1
camera_trigger_out_line: /PXI6259/ctr0
trigger_source: /PXI6259/PFI0
# Digital Laser Outputs
laser_port_switcher: PXI6733/port0/line1
laser_switch_state: False
camera:
hardware:
type: HamamatsuOrcaLightning #SyntheticCamera
serial_number: 000035
defect_correct_mode: 2.0
delay: 10 #ms
settle_down: 0.0 #ms
remote_focus_device:
hardware:
type: NI
channel: PXI6259/ao3
min: -0.5
max: 0.5
# Optotune EL-16-40-TC-VIS-5D-1-C
galvo:
-
hardware:
type: NI
channel: PXI6259/ao1
min: 0
max: 5
phase: 1.57079 # pi/2
filter_wheel:
hardware:
type: ASI #SyntheticFilterWheel
wheel_number: 2
port: COM8
baudrate: 115200
filter_wheel_delay: .030 # in seconds
available_filters:
BLU - FF01-442/42-32: 0
GFP - FF01-515/30-32: 1
RFP - FF01-595/31-32: 2
Far-Red - FF01-670/30-32: 3
Blocked1: 4
Empty: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
stage:
hardware:
-
type: ASI
serial_number: 123456789
port: COM8
baudrate: 115200
axes: [x, y, z, f] # Software
axes_mapping: [M, Y, X, Z] # M Shear axis mapping
#axes_mapping: [M, X, Y, Z] #testing y
#axes_mapping: [M, X, Z, Y] #testing Z
# axes_mapping: [Z, Y, X, M]
feedback_alignment: [90, 90, 90, 90]
-
name: stage
type: SyntheticStage
serial_number: 987654321
axes: [theta]
x_max: 0 # Swapped from Z
x_min: -22708.3 # Swapped from Z
y_max: 1361.3
y_min: -3496.3
z_max: 3521.9 # Swapped from X
z_min: -4551.1 # Swapped from X
f_max: 3233.0 #=m
f_min: -9382.0 #=m
theta_max: 360
theta_min: 0
external_trigger: /PXI6259/PFI1
# joystick_axes: [x, y, z, f]
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
N/A: 0
pixel_size:
N/A: 0.168
shutter:
hardware:
type: NI
channel: PXI6259/port0/line0
min: 0
max: 5
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 405
onoff:
hardware:
type: NI
channel: PXI6733/port0/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao0
min: 0
max: 5
type: LuxX
- wavelength: 488
onoff:
hardware:
type: NI
channel: PXI6733/port0/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao1
min: 0
max: 5
type: LuxX
- wavelength: 561
onoff:
hardware:
type: NI
channel: PXI6733/port0/line4
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao2
min: 0
max: 5
type: Obis
- wavelength: 642
onoff:
hardware:
type: NI
channel: PXI6733/port0/line5
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao3
min: 0
max: 5
type: LuxX
- wavelength: LED
power:
hardware:
type: NI
channel: PXI6733/ao4
min: 0
max: 5
Macroscale:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: PXI6259/port0/line1
camera_trigger_out_line: /PXI6259/ctr0
trigger_source: /PXI6259/PFI0
# Digital Laser Outputs
laser_port_switcher: PXI6733/port0/line1
laser_switch_state: True
camera:
hardware:
type: Photometrics #SyntheticCamera
serial_number: 1
camera_connection: PMPCIECam00
x_pixels: 5056.0
y_pixels: 2960.0
pixel_size_in_microns: 4.25
defect_correct_mode: 2.0
delay: 25 #8 #5.0
settle_down: 0.0 #ms
remote_focus_device:
hardware:
type: NI
channel: PXI6259/ao3
min: -0.5
max: 0.5
# Optotune EL-16-40-TC-VIS-5D-1-C
galvo:
-
hardware:
type: NI
channel: PXI6259/ao1
min: 0
max: 5
phase: 1.57079 # pi/2
filter_wheel:
hardware:
type: ASI #SyntheticFilterWheel
wheel_number: 2
port: COM8
baudrate: 115200
filter_wheel_delay: .030 # in seconds
available_filters:
BLU - FF01-442/42-32: 0
GFP - FF01-515/30-32: 1
RFP - FF01-595/31-32: 2
Far-Red - FF01-670/30-32: 3
Blocked1: 4
Empty: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
stage:
hardware:
-
name: stage
type: ASI
port: COM8
baudrate: 115200
serial_number: 123456789
axes: [x, y, z, f] #Software
# axes_mapping: [M, Y, X, Z]
axes_mapping: [M, Y, X, Z] #M shear
feedback_alignment: [90, 90, 90, 90]
-
name: stage
type: SyntheticStage
serial_number: 987654321
axes: [theta]
x_max: 0 # Swapped from Z
x_min: -22708.3 # Swapped from Z
y_max: 1361.3
y_min: -3496.3
z_max: 3521.9 # Swapped from X
z_min: -4651.1 # Swapped from X
f_max: 3233.0 #=m
f_min: -9382.0 #=m
theta_max: 360
theta_min: 0
external_trigger: /PXI6259/PFI1
# joystick_axes: [x, y, z, f]
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
N/A: 0
pixel_size:
N/A: 1.06
shutter:
hardware:
type: NI
channel: PXI6259/port0/line0
min: 0
max: 5
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 405
onoff:
hardware:
type: NI
channel: PXI6733/port0/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao0
min: 0
max: 5
type: LuxX
- wavelength: 488
onoff:
hardware:
type: NI
channel: PXI6733/port0/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao1
min: 0
max: 5
type: LuxX
- wavelength: 561
onoff:
hardware:
type: NI
channel: PXI6733/port0/line4
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao2
min: 0
max: 5
type: Obis
- wavelength: 642
onoff:
hardware:
type: NI
channel: PXI6733/port0/line5
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao3
min: 0
max: 5
type: LuxX
- wavelength: LED
power:
hardware:
type: NI
channel: PXI6733/ao4
min: 0
max: 5
gui:
channels:
count: 5
MCT-ASLM
(Top) Two different perspectives of the multiscale cleared tissue Axially Swept Light-Sheet Microscope (MCT-ASLM). (Bottom) A mouse lung imaged at 1x, 6x, and 37x magnification. Automatic event-driven switching between the low-resolution and high-resolution modes. Scale bars, 50 microns.
The Multiscale Cleared Tissue Axially Swept Light-Sheet Microscope (MCT-ASLM) is a sophisticated imaging system designed to investigate rare processes within large, chemically cleared multicellular samples. This advanced microscope incorporates dual imaging systems, termed the macroscale and nanoscale modules, which are uniquely aligned in a 4-axis geometry to optimize observation and analysis.
The macroscale module utilizes a single-sided illumination technique adapted from mesoSPIM, featuring a modified Nikon AF-S 50mm camera lens that projects light in a digitally scanned light-sheet format. This design minimizes shadows and enhances image clarity. Illumination control is achieved through an electro-tunable lens that synchronizes the laser beam’s movement with the rolling shutter of a CMOS camera, specifically a Hamamatsu Flash 4.0 V3. This setup allows for low numerical aperture axial light-sheet microscopy (ASLM) and supports variable magnification via an Olympus MVX-10 macro microscope for effective detection.
In contrast, the nanoscale module is designed for high-resolution imaging with a numerical aperture (NA) of 0.7 and uses multi-immersion objectives suitable for various solvents with refractive indices between 1.33 and 1.56. These objectives are engineered to be free from spherical and chromatic aberrations, and they maintain a significant working distance of 10mm. Aberration-free focusing is managed by a voice coil, and shadow reduction is enhanced with a resonant galvanometer that provides multidirectional illumination.
Central to its operation, the specimen is precisely positioned at the intersection of both imaging modules using high-precision translation and rotation stages that offer extensive travel distances (approximately 50-200 mm). This configuration enables rapid switching between macroscale and nanoscale imaging modes. The MCT-ASLM is adept at quickly producing detailed scans of large cleared tissues (up to 20 x 20 x 20 mm in size) at a resolution of 5 microns. Furthermore, it integrates advanced computer vision techniques to automatically identify biological features of interest and allows for detailed analysis with isotropic resolution up to 300 nm. A publication describing the MCT-ASLM system is currently in preparation.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.
Isotropic imaging across spatial scales with axially swept light-sheet microscopy
Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Omicron LightHUB Ultra with 488, 561, and 642 nm lasers. |
Stages |
PI L-509.20DG10, L-509.40DG10, L-509.20DG10, M-060.DG, M-406.4PD, PI P726.1CD |
Stage Controllers |
C-884, E-709 |
Cameras |
Hamamatsu Flash 4.0, Hamamatsu Fusion |
Filter Wheel |
Sutter Lambda 10-3 with 2x 32mm High-Speed Filter Wheels |
Remote Focusing Units |
Optotune Electrotunable Lens (EL-16-40-TC-VIS-5D-1-C) and Equipment Solutions LFA-2004 |
Data Acquisition Cards |
National Instruments PXIe-1073 chassis equipped with PXI6733 and PXI6259 |
Galvo |
Novanta CRS 4 KHz Resonant Galvo and Thorlabs GVS112 Linear Galvo |
Zoom |
Dynamixel MX-28R |
Other |
NA |
Configuration File
# Only one microscope can be active in the GUI at a time, but all microscopes will be accessible
microscopes:
Mesoscale:
daq:
hardware:
name: daq
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
sweep_time: 0.2
# triggers
master_trigger_out_line: PXI6259/port0/line1
camera_trigger_out_line: /PXI6259/ctr0
trigger_source: /PXI6259/PFI0
# Digital Laser Outputs
laser_port_switcher: PXI6733/port0/line0
laser_switch_state: False
camera:
hardware:
name: camera
type: HamamatsuOrca
serial_number: 302352
x_pixels: 2048.0
y_pixels: 2048.0
pixel_size_in_microns: 6.5
subsampling: [1, 2, 4]
sensor_mode: Normal # 12 for progressive, 1 for normal.
readout_direction: Top-to-Bottom # Top-to-Bottom', 'Bottom-to-Top'
lightsheet_rolling_shutter_width: 608
defect_correct_mode: 2.0
binning: 1x1
readout_speed: 0x7FFFFFFF
trigger_active: 1.0
trigger_mode: 1.0 # external light-sheet mode
trigger_polarity: 2.0 # positive pulse
trigger_source: 2.0 # 2 = external, 3 = software.
exposure_time: 20 # Use milliseconds throughout.
delay_percent: 10
pulse_percent: 1
line_interval: 0.000075
display_acquisition_subsampling: 4
average_frame_rate: 4.969
frames_to_average: 1
exposure_time_range:
min: 1
max: 1000
step: 1
flip_x: False
flip_y: False
x_pixels_step: 4
y_pixels_step: 4
x_pixels_min: 4
y_pixels_min: 4
remote_focus_device:
hardware:
name: daq
type: NI
channel: PXI6259/ao2
min: -5
max: 5
# Optotune EL-16-40-TC-VIS-5D-1-C
delay_percent: 7.5
ramp_rising_percent: 85
ramp_falling_percent: 2.5
amplitude: 0.7
offset: 2.3
smoothing: 0.0
galvo:
-
hardware:
name: daq
type: NI
channel: PXI6259/ao0
min: -5
max: 5
frequency: 99.9
amplitude: 2.5
offset: 0
duty_cycle: 50
phase: 1.57079 # pi/2
filter_wheel:
hardware:
name: filter_wheel
type: SutterFilterWheel
wheel_number: 1
port: COM2
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
Empty-Alignment: 5
GFP - FF01-515/30-32: 6
RFP - FF01-595/31-32: 7
Far-Red - BLP01-647R/31-32: 8
Blocked1: 4
Blocked2: 0
Blocked3: 1
Blocked4: 2
Blocked5: 3
Blocked6: 9
stage:
hardware:
name: stage
type: PI
serial_number: 119060508
axes: [x, y, z, theta, f]
controllername: C-884
stages: L-509.20DG10 L-509.40DG10 L-509.20DG10 M-060.DG M-406.4PD NOSTAGE
refmode: FRF FRF FRF FRF FRF FRF
x_max: 100000
x_min: -100000
y_max: 100000
y_min: -100000
z_max: 100000
z_min: -100000
f_max: 100000
f_min: 0
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
f_offset: 0
theta_offset: 0
flip_x: False
flip_y: False
flip_z: False
zoom:
hardware:
type: DynamixelZoom
servo_id: 1
port: COM9
baudrate: 1000000
position:
0.63x: 0
1x: 627
2x: 1711
3x: 2301
4x: 2710
5x: 3079
6x: 3383
pixel_size:
0.63x: 9.7
1x: 6.38
2x: 3.14
3x: 2.12
4x: 1.609
5x: 1.255
6x: 1.044
stage_positions:
BABB:
f:
0.63x: 67410
1x: 70775
2x: 72455
3x: 72710
4x: 72795
5x: 72850
6x: 72880
CUBIC:
f:
0.63x: 67410
1x: 70775
2x: 72455
3x: 72710
4x: 72795
5x: 72850
6x: 72880
shutter:
hardware:
type: NI
channel: PXI6259/port0/line0
min: 0
max: 5
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 488
onoff:
hardware:
type: NI
channel: PXI6733/port0/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao0
min: 0
max: 5
type: LuxX
- wavelength: 562
onoff:
hardware:
type: NI
channel: PXI6733/port0/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao1
min: 0
max: 5
type: Obis
- wavelength: 642
onoff:
hardware:
type: NI
channel: PXI6733/port0/line4
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao2
min: 0
max: 5
type: LuxX
Nanoscale:
daq:
hardware:
type: NI
# NI PCIe-1073 Chassis with PXI-6259 and PXI-6733 DAQ Boards.
# Sampling rate in Hz
sample_rate: 100000
# triggers
master_trigger_out_line: PXI6259/port0/line1
camera_trigger_out_line: /PXI6259/ctr0
trigger_source: /PXI6259/PFI0
# Digital Laser Outputs
laser_port_switcher: PXI6733/port0/line0
laser_switch_state: True
camera:
hardware:
type: HamamatsuOrca
serial_number: 500502
defect_correct_mode: 2.0
delay: 10 #ms
settle_down: 0.0 #ms
remote_focus_device:
hardware:
type: EquipmentSolutions #NI
channel: PXI6259/ao3
port: COM6
min: -5
max: 5
# waveform: trig_remote_focus_ramp
galvo:
-
hardware:
type: NI
channel: PXI6259/ao1
min: -5
max: 5
filter_wheel:
hardware:
type: SutterFilterWheel
wheel_number: 2
port: COM2
baudrate: 9600
filter_wheel_delay: .030 # in seconds
available_filters:
Empty-Alignment: 0
GFP - FF01-515/30-32: 1
RFP - FF01-595/31-32: 2
Far-Red - BLP01-647R/31-32: 3
Blocked1: 4
Blocked2: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
stage:
hardware:
-
type: PI
serial_number: 119060508
axes: [x, y, z, theta]
controllername: C-884
stages: L-509.20DG10 L-509.40DG10 L-509.20DG10 M-060.DG M-406.4PD NOSTAGE
refmode: FRF FRF FRF FRF FRF FRF
-
type: PI
serial_number: 0116049747
axes: [f]
controllername: E-709
stages: P-726.1CD
refmode: ATZ
# -
# name: stage2
# type: MCL
# serial_number: 4011
# axes: [f]
x_max: 100000
x_min: -100000
y_max: 100000
y_min: -100000
z_max: 100000
z_min: -100000
f_max: 100000
f_min: 0
theta_max: 360
theta_min: 0
x_offset: 500 # -1000
y_offset: 300 # -70
z_offset: -18396 # -17842
# x_offset: 0
# y_offset: 0
# z_offset: 0
f_offset: 0
theta_offset: 0
zoom:
position:
N/A: 0
pixel_size:
N/A: 0.167
shutter:
hardware:
type: NI
channel: PXI6259/port2/line0
lasers:
# Omicron LightHub Ultra
# 488 and 640 are LuxX+ Lasers
# 561 is a Coherent OBIS Laser
# Digital Laser Outputs
- wavelength: 488
onoff:
hardware:
type: NI
channel: PXI6733/port0/line2
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao0
min: 0
max: 5
type: LuxX
- wavelength: 562
onoff:
hardware:
type: NI
channel: PXI6733/port0/line3
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao1
min: 0
max: 5
type: Obis
- wavelength: 642
onoff:
hardware:
type: NI
channel: PXI6733/port0/line4
min: 0
max: 5
power:
hardware:
type: NI
channel: PXI6733/ao2
min: 0
max: 5
type: LuxX
gui:
channels:
count: 5
Spectral TIRF
Images of the custom-built ring-TIRF microscope.
This custom-built ring-TIRF microscope is designed for spectrally multiplexed imaging of intracellular targets, utilizing a combination of spectral discrimination and linear unmixing techniques. It features a comprehensive 7-line laser launch, emitting at 445 nm, 488 nm, 514 nm, 532 nm, 561 nm, 594 nm, and 640 nm. Ring-TIRF provides uniform illumination across the field of view and allows for rapid, wavelength-dependent switching between epi-illumination and TIRF modes. This microscope supports FRET detection, expanding its utility in live cell imaging applications. Imaging is executed using an Olympus NA 1.7 TIRF lens (APON100XHOTIRF) paired with two Hamamatsu Flash 4.0 v3 cameras, enabling high-resolution and high-sensitivity imaging. A publication describing the spectral TIRF system is currently in preparation.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Omicron LightHUB Ultra with 405, 457, 488, 514, 532, 561, and 642 nm lasers. |
Stages |
ASI LS-50 linear stage and MS-2000 XY stage. |
Stage Controllers |
ASI Tiger Controller |
Cameras |
2x Hamamatsu Flash 4.0 |
Filter Wheel |
2x ASI 6-Position 32 mm Filter Wheels, and 1x motorized ASI dichroic slider. |
Remote Focusing Units |
N/A |
Data Acquisition Cards |
National Instruments PCIe-1073 chassis equipped with PCIe-6259 and PCIe-6738 |
Galvo |
2x Novanta Linear Galvos. |
Zoom |
N/A |
Other |
NA |
Configuration File
microscopes:
SpectralTIRF_lt538:
daq:
hardware:
name: daq
type: NI
sample_rate: 100000
sweep_time: 0.2
master_trigger_out_line: PCIE6738/port0/line1
camera_trigger_out_line: /PCIE6738/ctr0
trigger_source: /PCIE6738/PFI0
laser_port_switcher: PCIE6738/port0/line0
laser_switch_state: False
# dichroic:
# hardware:
# name: ASI
# type: ASI #synthetic #ASI
# port: COM17
# axes: [S]
# baudrate: 115200
# available_dichroics:
# 510LP: 0
# 570LP: 1
# 640LP: 2
camera:
hardware:
name: camera
type: HamamatsuOrca
serial_number: 003055
x_pixels: 2048.0
y_pixels: 2048.0
pixel_size_in_microns: 6.5
subsampling: [1, 2, 4]
sensor_mode: Normal # 12 for progressive, 1 for normal. Normal/Light-Sheet
readout_direction: Top-to-Bottom # Top-to-Bottom', 'Bottom-to-Top'
lightsheet_rolling_shutter_width: 608
defect_correct_mode: 2.0
binning: 1x1
readout_speed: 1.0
trigger_active: 1.0
trigger_mode: 1.0 # external light-sheet mode
trigger_polarity: 2.0 # positive pulse
trigger_source: 2.0 # 2 = external, 3 = software.
exposure_time: 20 # Use milliseconds throughout.
delay_percent: 10
pulse_percent: 1
line_interval: 0.000075
display_acquisition_subsampling: 4
average_frame_rate: 4.969
frames_to_average: 1
exposure_time_range:
min: 1
max: 1000
step: 1
x_pixels_step: 4
y_pixels_step: 4
x_pixels_min: 4
y_pixels_min: 4
remote_focus_device:
hardware:
name: daq
type: NI
channel: PCIE6738/ao2
min: 0
max: 5
delay_percent: 7.5
ramp_rising_percent: 85
ramp_falling_percent: 2.5
amplitude: 0.7
offset: 2.3
galvo:
-
hardware:
name: daq
type: NI
channel: PCIE6738/ao2 #galvo-x
min: -5
max: 5
waveform: sine
frequency: 99.9
period: 10
amplitude: 2.5
offset: 0
duty_cycle: 50
phase: 0
-
hardware:
name: daq
type: NI
channel: PCIE6738/ao3 #galvo-y
min: -5
max: 5
waveform: sine
frequency: 99.9
period: 10
amplitude: 2.5
offset: 0
duty_cycle: 50
phase: 90
filter_wheel:
-
hardware:
name: emission
type: ASI
wheel_number: 0
port: COM17
baudrate: 115200
filter_wheel_delay: .030 # in seconds
available_filters:
Empty 1: 0
482/35: 1
520/15: 2
NF03-488E: 3
Blocked2: 4
Blocked3: 5
Empty 2: 6
Blocked 4: 7
Blocked 5: 8
Blocked 6: 9
-
hardware:
name: emission
type: ASI
wheel_number: 1
port: COM17
baudrate: 115200
filter_wheel_delay: .030 # in seconds
available_filters:
Empty 1: 0
575/15: 1
647/57: 2
Blocked: 3
593/lp: 4
578/21: 5
Empty 2: 6
Blocked 4: 7
Blocked 5: 8
Blocked 6: 9
stage:
hardware:
-
name: ASI
type: ASI
port: COM17
baudrate: 115200
serial_number: 119060508
axes: [x, y, z]
axes_mapping: [X, Y, Z]
volts_per_micron: None
axes_channels: None
max: None
min: None
-
name: Synthetic
type: SyntheticStage
serial_number: 119060508
axes: [theta, f]
axes_mapping: [theta, f]
volts_per_micron: None
axes_channels: None
max: None
min: None
x_max: 50000
x_min: -50000
y_max: 50000
y_min: -50000
z_max: 50000
z_min: -50000
f_max: 50000
f_min: 0
theta_max: 360
theta_min: 0
x_step: 500
y_step: 500
z_step: 500
theta_step: 30
f_step: 500
velocity: 1000
zoom:
hardware:
name: zoom
type: synthetic
servo_id: 1
position:
110x: 0
pixel_size:
110x: 0.059
shutter:
hardware:
name: daq
type: synthetic
channel: PCIE6738/port0/line0
min: 0
max: 5
lasers:
- wavelength: 405
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/port1/line1 #38/35
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao21
min: 0
max: 5
type: Obis
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 445
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao4
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao5
min: 0
max: 5
type: LuxX
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 488
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao6
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao7
min: 0
max: 5
type: LuxX
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 514
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao8
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao9
min: 0
max: 5
type: LuxX
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 532
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao10
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao11
min: 0
max: 5
type: LuxX
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 562
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao12
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao13
min: 0
max: 5
type: Obis
index: 1
delay_percent: 10
pulse_percent: 87
- wavelength: 592
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao14
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao15
min: 0
max: 5
type: LuxX
index: 0
delay_percent: 10
pulse_percent: 87
- wavelength: 642
onoff:
hardware:
name: daq
type: NI
channel: PCIE6738/ao16
min: 0
max: 5
power:
hardware:
name: daq
type: NI
channel: PCIE6738/ao17
min: 0
max: 5
type: LuxX
index: 2
delay_percent: 10
pulse_percent: 87
gui:
channels:
count: 5
laser_power:
min: 0
max: 100
step: 10
exposure_time:
min: 1
max: 1000
step: 5
interval_time:
min: 0
max: 1000
step: 5
stack_acquisition:
step_size:
min: 0.200
max: 1000
step: 0.1
start_pos:
min: -5000
max: 5000
step: 1
end_pos:
min: -5000
max: 10000
step: 1
timepoint:
timepoints:
min: 1
max: 1000
step: 1
stack_pause:
min: 0
max: 1000
step: 1
Robotic MesoSPIM
A CAD image of the single sided variant of the benchtop mesoSPIM equipped with a Meca500 robotic arm and a Thorlabs rotary stage for automatic sample loading and unloading.
This single-sided illumination benchtop mesoSPIM features an Oxxius L4Cc laser launch equipped with 488, 561, and 638 nm lasers, Applied Scientific Instrumentation stages for positioning in X, Y, Z, Theta, and Focus. The microscope uses an XLFLUOR4X/340 NA 0.28 illumination objective, which is well-suited for larger field views and deeper penetration into the samples. Imaging is performed with a Hamamatsu Flash 4.0 camera and an 8-position emission filter wheel. A galvanometer provides digitally scanned illumination and an electrotunable lens facilitates scanning of the illumination beam along its propagation direction, enabling Axially Swept Light-Sheet Imaging. Additionally, the setup includes a Meca500 robotic arm, which automates the loading and unloading of samples, significantly enhancing experimental throughput. A publication describing the robotic mesoSPIM system is currently in preparation.
Instrument Location:
The University of Texas Southwestern Medical Center, Dallas, TX
Relevant Publications:
Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples
The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue
Technical Information
Equipment |
Description |
|---|---|
Lasers |
Oxxius L4Cc-CSB-1211-488-561-638-300 |
Stages |
ASI stages for benchtop mesoSPIM. Provides control in X, Y, Z, Theta, and F. |
Stage Controllers |
ASI Tiger Controller |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
ASI 8-Position 25 mm Filter Wheel |
Remote Focusing Units |
Optotune Electrotunable Lens (EL-16-40-TC-VIS-5D-1-C) |
Data Acquisition Cards |
National Instruments PCIe-6723 |
Galvo |
Thorlabs GVS112 Linear Galvo |
Zoom |
N/A |
Other |
Meca500 robotic arm for sample loading and unloading. |
BioFrontiers OPM
The BioFrontiers Institute, a designated Beckman Center for Advanced Light-Sheet Microscopy and Data Science, is developing an Oblique Plane Microscopy (OPM) system. This system features a high numerical aperture (NA) water dipping objective specifically designed for cutting-edge research on stem cell-derived organoids and live imaging of mammalian embryonic neural tube closure. Stem cell-derived organoids are increasingly recognized as superior in vitro models of human organ development and disease. They offer significant potential in the realms of drug discovery and tissue repair, owing to their ability to mimic the complex architecture and functionality of human tissues. These organoids are produced through self-organization processes that replicate the intricate structural and functional characteristics of natural organs, making them invaluable in medical and biological research. Additionally, the OPM system will be instrumental in studying neural tube development—an embryonic structure that eventually forms the brain and spinal cord. Understanding the mechanisms of neural tube closure is crucial, as its failure leads to neural tube defects such as spina bifida.
Instrument Location:
The University of Colorado, Boulder, CO
Relevant Publications:
Technical Information
Equipment |
Description |
|---|---|
Lasers |
3i LaserStack with 405, 488, 561, and 642 nm lasers. |
Stages |
ASI FTP-2000 with MS-2000 XY stage, and a Galvo for acquisition of z-stacks. |
Stage Controllers |
ASI Tiger Controller |
Cameras |
Hamamatsu Flash 4.0 |
Filter Wheel |
ASI 8-Position 25 mm Filter Wheel |
Remote Focusing Units |
N/A |
Data Acquisition Cards |
National Instruments PCIe-6723 |
Galvo |
Thorlabs GVS112 Linear Galvo |
Zoom |
N/A |
Other |
NA |
Configuration File
microscopes:
OPM:
daq:
hardware:
type: NI
sample_rate: 100000
master_trigger_out_line: PXI6723/port0/line1
camera_trigger_out_line: /PXI6723/ctr0
trigger_source: /PXI6723/PFI0
laser_port_switcher: PXI6723/port0/line0
laser_switch_state: False
camera:
hardware:
type: HamamatsuOrca
serial_number: 000646
defect_correct_mode: 2.0
delay: 10.0 #ms
settle_down: 0.0 #ms
flip_x: False
flip_y: False
remote_focus_device:
hardware:
type: SyntheticRemoteFocus
channel: PXI6723/ao2
min: 0
max: 5
galvo:
-
hardware:
type: NI
channel: PXI6723/ao0
min: -10
max: 10
waveform: sine
phase: 1.57079
filter_wheel:
hardware:
type: ASI
wheel_number: 1
port: COM13
baudrate: 115200
filter_wheel_delay: .030
available_filters:
Empty-Alignment: 0
GFP - FF01-515/30-32: 1
RFP - FF01-595/31-32: 2
Far-Red - BLP01-647R/31-32: 3
Blocked1: 4
Blocked2: 5
Blocked3: 6
Blocked4: 7
Blocked5: 8
Blocked6: 9
stage:
hardware:
-
type: ASI
serial_number: 123456789
axes: [x, y, f]
axes_mapping: [X, Y, Z]
volts_per_micron: None
max: None
min: None
port: COM13
baudrate: 115200
timeout: 0.25
-
type: GalvoNIStage
serial_number: 987654321
axes: [z]
axes_mapping: [ PXI6723/ao1 ]
volts_per_micron: 0.05*x
distance_threshold: None
settle_duration_ms: 20 #ms
max: 10
min: -10
-
type: SyntheticStage
serial_number: 123
axes: [theta]
axes_mapping: [theta]
max: 360
min: 0
joystick_axes: [x, y, z]
x_max: 100000
x_min: -100000
y_max: 100000
y_min: -100000
z_max: 100000
z_min: -100000
f_max: 100000
f_min: -100000
theta_max: 360
theta_min: 0
x_offset: 0
y_offset: 0
z_offset: 0
theta_offset: 0
f_offset: 0
flip_x: False
flip_y: False
flip_z: False
zoom:
hardware:
type: SyntheticZoom
servo_id: 1
position:
0.63x: 0
1x: 627
2x: 1711
3x: 2301
4x: 2710
5x: 3079
6x: 3383
pixel_size:
0.63x: 9.7
1x: 6.38
2x: 3.14
3x: 2.12
4x: 1.609
5x: 1.255
6x: 1.044
stage_positions:
BABB:
f:
0.63x: 0
1x: 1
2x: 2
3x: 3
4x: 4
5x: 5
6x: 6
shutter:
hardware:
type: SyntheticShutter #NI
channel: PXI6723/port0/line0
min: 0
max: 5
lasers:
- wavelength: 405
onoff:
hardware:
type: NI
channel: PCI6321/port0/line3
min: 0
max: 5
power: #analog
hardware:
type: SyntheticLaser
channel: PCI6321/ao1
min: 0
max: 5
type: LuxX
- wavelength: 488
onoff: #digital
hardware:
type: NI
channel: PCI6321/port0/line5
min: 0
max: 5
power: #analog
hardware:
type: SyntheticLaser
channel: PCI6321/ao0
min: 0
max: 5
type: LuxX
- wavelength: 561
onoff:
hardware:
type: NI
channel: PCI6321/port0/line7
min: 0
max: 5
power: #analog
hardware:
type: SyntheticLaser
channel: PCI6321/ao1
min: 0
max: 5
type: Obis
- wavelength: 640
onoff: #digital
hardware:
type: NI
channel: PCI6321/port0/line1
min: 0
max: 5
power: #analog
hardware:
type: SyntheticLaser
channel: PCI6321/ao2
min: 0
max: 5
type: LuxX
gui:
channels:
count: 5
Shepherd Lab MesoSPIM
More coming soon.
Instrument Location:
Arizona State University, Tempe, AZ
Relevant Publications:
The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.
Isotropic imaging across spatial scales with axially swept light-sheet microscopy
Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.
Technical Information
Equipment |
Description |
|---|---|
Lasers |
TBD |
Stages |
TBD |
Stage Controllers |
TBD |
Cameras |
TBD |
Filter Wheel |
TBD |
Remote Focusing Units |
TBD |
Data Acquisition Cards |
TBD |
Galvo |
TBD |
Zoom |
TBD |
Other |
TBD |
Shepherd Lab OPM
More coming soon.
Instrument Location:
Arizona State University, Tempe, AZ
Relevant Publications:
Technical Information
Equipment |
Description |
|---|---|
Lasers |
TBD |
Stages |
TBD |
Stage Controllers |
TBD |
Cameras |
TBD |
Filter Wheel |
TBD |
Remote Focusing Units |
TBD |
Data Acquisition Cards |
TBD |
Galvo |
TBD |
Zoom |
TBD |
Other |
TBD |