Longitudinal Intravital Imaging Through Clear Silicone Windows

Summary

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Abstract

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse’s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time—up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

Introduction

Intravital microscopy (IVM), the imaging of tissues in anesthetized animals, offers insights into the dynamics of physiological and pathological events at cellular resolution in intact tissues. The applications of this technique vary widely, but IVM has been instrumental in the cancer biology field to help elucidate how cancer cells invade tissues and metastasize, interact with the surrounding microenvironment, and respond to drugs^1,2,3. In addition, IVM has been key to advancing the understanding of the complex mechanisms governing immune responses by providing insights comp....

Protocol

All procedures described were performed in accordance with the Cold Spring Harbor Laboratory Surgical Guidelines and had been approved by the Institutional Animal Care and Use Committee at Cold Spring Harbor Laboratory.

1. Casting the silicone window

1. Prepare the silicone polymer (PDMS) by mixing the base elastomer and curing agent in a 10:1 (v/v) ratio.
2. Cast a window by depositing a small quantity of PDMS on a sterile, smooth surface and adjust the volum.......

Discussion

Intravital imaging windows are important tools for directly visualizing physiological and pathological processes at cellular resolution as they unfold over time. The novel procedure described for casting and inserting flexible, silicone imaging windows in mice overcomes some of the most common issues with currently used imaging windows (exudate, breaking, and interference with normal mobility), provides additional safety for the mouse, and increases the accessibility of this technique.

The mos.......

Materials

Name	Company	Catalog Number	Comments
3M Medipore Soft Cloth Surgical Tape	3M	70200770819
Silk suture 4-0 PERMA HAND BLACK 1 x 18" RB-2	Ethicon	N267H
ACTB-ECFP mice	Jackson Laboratory	22974
AEC Substrate Kit, Peroxidase (HRP), (3-amino-9-ethylcarbazole)	Vector Laboratories	SK-4200
Alcohol swabs	BD	326895
Anesthesia system	Molecular Imaging Products Co.
Acqknowledge software and sensors	BIOPAC	ACK100W, ACK100M, TSD110
Betadine spray	LORIS	109-08
c-fms-EGFP (MacGreen) mice	Jackson Laboratory	18549
C57BL/6J mice	Jackson Laboratory	664
CD45 Monoclonal Antibody (30-F11)	Invitrogen	14-0451-82
CD68 Antibody	Abcam	ab125212
Curity gauze sponges	Covidien
Donkey Anti-Goat IgG H&L (HRP)	Abcam	ab6885
Donkey Anti-Rabbit IgG H&L (HRP)	Abcam	ab97064
Donkey Anti-Rat IgG H&L (HRP)	Abcam	ab102182
Dow SYLGARD 184 Silicone Encapsulant Clear	Electron Microscopy Sciences	24236-10	Two-part, 10:1 mixing ratio
Round Cover Glass, 8mm Diameter, #1.5 Thickness	Electron Microscopy Sciences	72296-08
Ender-3 Pro 3D printer	Shenzhen Creality 3D Technology Co., LTD
Far Infrared Heated blanket	Kent Scientific	RT-0520
Fc Receptor Blocker	Innovex Biosciences	NB309
Fiji imaging processing package			https://imagej.net/software/fiji/
FluoroSpheres carboxylate, 0.04µm, yellow-green (505/515)	Invitrogen	F8795
Gating system:	BIOPAC Systems Inc.		The components together allow monitoring mouse vitals during imaging and gating image acquisition on mouse respiration. All were acquired from BIOPAC systems.
Acqknowledge software		ACK100W, ACK100M
Diff. Amp. Module, C Series		DA100C
Dual Gating Sys small animal		DTU200
MP160 for Windows - Analysis system		MP160WSW
MouseOx Plus 120V		MOX-120V;015000
Pressure Pad		TSD110
Gelfoam	Pfizer	9031508	Absorbable gelatin sponge
Hardened fine scissors	Fine Science Tools	14090-11	Two pairs; stainless steel, sharp-sharp tips, straight tip, 26 mm cutting edge, 11 cm length
Human/Mouse Myeloperoxidase/MPO Antibody	R&D Systems	AF3667
Hot bead sterilizer	Fine Science Tools	18000-45	Turn on approximately 30 min before use; sterilize tools at >200 °C for 30 s
Imaris	Bitplane	www.bitplane.com
Immersion medium Immersol W 2010	Zeiss	444969-0000-000
Insulin Syringes with BD Ultra-Fine needle 6mm x 31G 1 mL/cc	BD	324912
Isoflurane (Fluriso)	VetOne	502017
Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight® 594	Vector Laboratories	DL-1177-1
LysM-eGFP mice	www.mmrrc.org	012039-MU
Micro dissecting forceps	Roboz	RS-5135	Serrated, slight curve, 0.8 mm tip width; 4" length
Micro dissecting forceps	Roboz	RS-5153	1 x 2 teeth, slight curve, 0.8 mm tip width, 4" length
MTS MiniBionix II 808	MTS Systems		Servohydraulic material testing machine
Neutrophil Elastase 680 FAST probe	PerkinElmer	NEV11169
Nitrogen	General Welding Supply Corp.
Oxygen	General Welding Supply Corp.
Polylactic acid filament	Hatchbox		1.75 mm diameter
ProLong Diamond Antifade Mountant	Invitrogen	P36970
Puralube ophthalmic ointment	Dechra	NDC17033-211-38
Reflex 7 wound clips	Roboz Surgical	RS-9255
Stainless steel grid	Fotofab		One grid is 0.200 inches in diameter, with a total of 52 individual grid squares that are 0.016 x 0.016 inches. There is 0.003 inches of space between each square.
Surface Treated SterileTissue Culture Plates	Fisher Scientific	FB012929	Lid used as curing surface for imaging windows
TriM Scope Multiphoton Microscope	LaVision BioTec		Imaging was done on an upright 2-photon microscope (Trimscope, LaVision BioTec) equipped with two Ti:Sapphire lasers (Mai Tai and InSight, Spectra-Physics) and an optical parametric oscillator. The following Longpass Dichroic Beamsplitters (Chroma) were used to direct the signal towards four photomultipler tubes: T560LP T665LPXXR T495lxpr
Vetbond	3M	70200742529
VWR micro cover glass	VWR	48404-453