Selective Plane Illumination Microscopy Setup for Volumetric Imaging

Kyiv, Ukraine

Setup based on open source projects.

Design notes

Tip

Read more about the SPIM principles:

• Selective Plane Illumination Microscopy
• openSPIM project
• Selective plane illumination microscopy techniques in developmental biology
• Using tissue clearing and light sheet fluorescence microscopy for the three-dimensional analysis of sensory and sympathetic nerve endings that innervate bone and dental tissue of mice

Gaussian litgh-sheet

Based on Olarte et al., 2018 and Power and Huisken, 2017.

Schematic of the illumination and detection arm in a SPIM (Olarte et al., 2018)

Illumination arm

Light-sheet thickness (axial sectioning resolution):

$$l_z = 2w_0 = 2 \frac{n \cdot \lambda_{ex.}}{\pi \cdot NA_{ex.}}$$

Light-sheet length (field of view/FOV):

$$l_x = 2z_r = 2 \frac{\pi \cdot w_0^2}{\lambda_{ex.}} = 2 \frac{n^2 \cdot \lambda_{ex.}}{\pi \cdot NA_{ex.}^2}$$

Detection arm

Lateral resolution (Rayleigh criterion):

$$d_{xy} = \frac{0.61 \cdot \lambda_{det.}}{NA_{det.}}$$

Axial resolution (depth of field/DOF):

$$d_z = \frac{n \cdot \lambda_{det.}}{NA_{det.}^2}$$

Illumination light path parameters

Samples and desired characteristics

NB: is 10x NA 0.25 FN 22 enough?

Sciatic nerve

• Nerve diameter: 1.5-2 mm
• Nerve length: 5-20 mm
• A-fiber diameter: 1.5 - 10 um
• C-fiber diameter: 0.5 - 2 um
• Minimal vessel diameter

Scaffolds

In progress

Hardware

• ESP32 with Arduino32bitBoards device adapter
• OXLaser 505 nm, 520 nm, and 650 nm
• Basler Ace 2R Pro

Objectives

Model	Olympus PlaN	Olympus UPlanFl Ph. 1
Magnification (M)	10x	10x
Numerical aperture (NA)	0.25	0.3
Working distance (WD)	10.6 mm	10 mm
Focal distance (F)	18 mm	18 mm
Back focal plane distance (BFP)	N/A	-19.1 mm
Field number (FN)	22	26.5 mm
Lateral resolution	1.34 μm	1.12 μm
Parafocal distance (PD)	45 mm	65 mm
Exit pupil diameter (EP)	9 mm	10.8 mm

Cameras

Model	FLIR Grasshopper 3	Basler Ace 2R
ID	GS3-U3-23S6M-C	a2A5320-23umPRO
Sensor	Sony IMX174 (CMOS)	Sony IMX542 (CMOS)
Sensor size	11.25x7.03 mm (1/1.2")	14.58x8.31 mm (1.1")
Sensor diagonal	13.4 mm	16.78 mm
Pixels (HxV)	1920x1200 (2.3 Mpx)	5320x3032 (16.1 Mpx)
Pixel size	5.86x5.85 μm	2.74x2.74 μm
Frame rate	up to 162 fps	up to 24 fps

FLIR Grasshopper 3 GPIO pinout

Basler Ace 2R GPIO pinout

Software

All control is provided with Micro-Manager and pymmcore-plus library with napari-micromanager GUI.

Configuration

Component	Desctiption	Micro-manager adapter	Port (laptop)	Note
Camera	FLIR Grasshopper3 USB3	Point Grey Research	USB 3.0 (COM7)
Stage	Sutter Instruments MPC-200	CustomArduino (MarzhauserLStep Z-stage)
Lasers	Laser control wtih TTL	Arduino	COM6	Change ArduMM version in sketch to 2

License

This open-source project is released under the CERN Open Hardware License. Our aims are to promote open scientific hardware development and to share our engineering solutions.