SPIM for DoMB

Selective Plane Illumination Microscope based on adaptations and modifications of original openUC2 and OpenFlexure Block Stage projects.

Department of Molecular Biophysics, Bogomoletz Institute of Physiology of NAS of Ukraine (Kyiv, Ukraine).

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L-SPIM system overview

Microscope configuration

The microscope consists of three main parts: (1) excitation light path with a light source, light source control board, and light sheet collimation system, (2) sample handling part which includes a sample chamber with modified OpenFlexure Block Stage and side illumination system, and (3) detection light path with emission filter, tube lens, and industrial-grade CMOS camera (FLIR Grasshopper 3 in our case). A solid yellow line indicates the excitation and detection light path, and a dashed yellow line indicates the brightfield side-illumination light path.

The whole setup is based on on-shelf parts and low-cost solutions with minimal additional modifications. For all 3D-printed parts, parametric openSCAD models were provided for flexible modification and customization without a great entry threshold.

This system is under strong development, so check for future updates.

First results

Light sheet configuration

Light sheet top view	Light sheet side view	Light sheet focus point view

Z-stack and reconstruction of whole mount staining of vessels endothelial cells in the sciatic nerve

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

Construction notes

General materials and parts needed for printing and assembling:

Part	#	Cost/unit	Link
Optical breadboard 30x45 cm	1x	$135	AliExpress
M3x12 DIN912 screw	~200x	~$8 / 200	Metalvis
M6x12 DIN912 screw	~30x	~$6 / 30	Metalvis
M6x12 DIN912 screw	~10x	~$1 / 10	Metalvis
M5x8 DIN914 screw	~150x	~$5 / 150	Metalvis
1.75 mm PLA filament (750 g/pack)	2x	$11	Monofilament
1.75 mm ABS+ filament (750 g/pack)	1.5x	$10.85	Monofilament
Total		~$200

1. Excitation light path

1. RGB lasers module and lasers trigger board
2. ~12x beam expander: iPhone 4S lens F3.85 and F50 lens
3. Cylinrical lens F30
4. 45° kinematic mirror
5. Focusing lens F50
6. Projection objective 10x NA 0.25

Part	#	Cost/unit	Link
RGB lasers module	1x	$23
iPhone 4S lens F3.85	1x	-
F50 D30 lens	2x	$0.68	AliExpress
F30 20x22 mm cylindrical lens	1x	$10.54	AliExpress
Fron surface mirror 30x30 mm	1x	$1.46	AliExpress
10x NA 0.25 infinity corrected objective	1x	$15.54	AliExpress
Total		~$52

2. Sample handling

1. White LED 3.3V 10W and 45° kinematic mirror
2. OpenFlexure Block Stage with sample holder
3. Sample chamber

Part	#	Cost/unit	Link
LED and power cube	1x	$12.68
Fron surface mirror 30x30 mm	1x	$1.46	AliExpress
Total		~$14

3. Detection light path

1. Objective 4x NA 0.1
2. Emission filter holder
3. Tube lens ~F180
4. Light protection enclosure
5. CMOS camera and C-mount cube

Part	#	Cost/unit	Link
4x NA 0.1 infinity corrected objective	1x	$13.25	AliExpress
25 mm emission filter	1x	discontinued old filter set, but new >$300
F~180 D40 lens	1x	from old stereomicroscope
FLIR Grasshopper 3	1x	$1179	Edmund Optics
Total		~$1195

Important

The cost of the entire microscope, excluding the CMOS camera and emission filters set, was less than $300. A possible alternative for expensive emission filters may be industrial-grade filters for multimedia projector systems, but with a minimal selection of possible passbands. I also recommend paying attention to other manufacturers of industrial monochrome cameras: Allied Vision, Basler, Daheng.

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Theoretical notes

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):

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

Samples and desired characteristics

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

NB: is 10x NA 0.3 FN 22 enough?

Relation of components and parameters

Objective

Model	Olympus UPlanFl Ph. 1
Magnification (M)	10x
Numerical aperture (NA)	0.3
Working distance (WD)	10 mm
Focal distance (F)	18 mm
Back focal plane distance (BFP)	-19.1 mm
Field number (FN)	26.5 mm
Lateral resolution	1.12 um
Parafocal distance (PD)	65 mm
Exit pupil diameter (EP)	10.8 mm

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T-SPIM

Here will be construction notes for the next project, T-SPIM extension of DoMB-SPIM.

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Cubes and modules

OpenFlexure Block Stage for SPIM

Active development of OpenFlexure Block Stage adaptation continues on a separate repository.

Block Stage side view

C-mount adapter

Parametric model of a cube insert with C-mount thread (c-mount_insert_v0.scad).

Model
Printed long insert
Installed insert

LED and power cube V0

Parametric model of a cube insert with two LED dimmers and 12V output with type-C Power Delivery input (led_power_insert_v0.scad).

Model

Part	#	Cost/unit	Link
LED dimmer	2x	$2.72	AliExpress
PD decoy module 12V	1x	$0.44	AliExpress
Mini560 Pro Step Down to 3.3V	1x	$0.81	AliExpress
White LED 10W 3.3V (XML2)	1x	$1.29	AliExpress
460 nm LED 3W 3.3V	1x	$0.2	AliExpress
Acrilic LED lens, F22 D20.8	2x	$1.35	AliExpress
XT30 connector	3x	$0.6	arduino.ua
Total		$12.68

Assembled power cube
Power cube with two LED cubes

White 10W LED cube with radiator from northbridge

Front view	Back view

25 mm filter holder V1

Sliding holder for standard 25 mm optical filters. The storage box for filter holders has mounting holes for standard M6 25mm spacing optical board.

Model

Assembled filter cube	Filter cube with filter holders and holders storage box

Sample chamber V0

Sample chamber with three glass walls, as chamber windows was used 18 mm diameter 0.18 mm thickness coverslips mounted on silicone sealant.

Model	Assembled chamber

Servo stage V1

The holder was adapted to install a plastic photometric cuvette for sample mounting.

Model Includes openSCAD library "Bibliothek für Evolventen-Zahnräder, Schnecken und Zahnstangen" (gear.scad)

Assembled cube

Top view	Side view

5X Laser beam expander V0

Model

Part	#	Cost/unit	Link
Acrilic LED lens, F22 D20.8	1x	$1.35	AliExpress
iPhone 4S lens, F3.85	1x	-

RGB lasers module V0

Module for mounting and control with Arduino of the RGB laser LED module.

Part	#	Cost/unit	Link
RGB laser module	1x	$19.5	AliExpress
Arduino Nano compatible board	1x	$2.13	AliExpress
Arduino Nano expansion board	1x	$0.94	AliExpress
PD decoy module 12V	1x	$0.44	AliExpress
Total		$23

RGB laser trigger board modification

The original diode bridge on the RBG laser trigger board (circled in red) was replaced with two jumpers for the DC power supply only, and the original trigger connectors were replaced with 2.54 mm pin headers.

Original board	Modified board

Nano expansion board modification

The expansion board's original DC connector (circled in red) was replaced with a PD trigger module for power supply with a power delivery source (AC adapter, power bank, etc.).

Original board (top)	Modified board (top)

Arduino pin 2 was pulled up with a 1K resistor to the 5V (circled in red) for connection to the passive trigger output of the Point Grey Grasshopper 3 camera (trigger output - pin 2, gnd - pin 6). The male XT30 connector for an optional power supply from the LED/power cube was soldered to the power input.

Original board (bottom)	Modified board (bottom)

Assembled control module

Lasers powered through an arduino Vin pin.

TTL inputs of the RGB laser trigger board was connected to the next arduino pins:

• 650 nm - 8
• 520 nm - 9
• 450 nm - 10

Trigger board holder	Assembled module

For arduino operating details see Step-by-step to controlling multiple light sources with an Arduino.

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Software

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

Configuration

Component	Desctiption	Micro-manager adapter	Note
Camera	FLIR Grasshopper3 USB3	Point Grey Research
Stage	Servo stage	CustomArduino (MarzhauserLStep Z-stage)	Set AnswerTimeout to 5000.000
Lasers	RGB laser control wtih TTL	Arduino	Change MM Ardu version in sketch to 2

Useful links

• Grasshopper3 USB 3.1 manual, PDF
• Step-by-step to controlling multiple light sources with an Arduino for sequenceable MDA by Guillaume Witz & Thomas Julou