Imaging windows for long-term intravital imaging

Figures & data

Figure 1. Intravital microscopy of tumors through imaging windows. (A) A 3D reconstruction of a murine orthotopic glioma (GL261-H2B Dendra2). The time-series show single cells migrating along the vessels. Nuclear localization of the photoprotein allows accurate single cell tracking. Green: tumor cell nuclei; Red: Dextran-labeled vasculature. Scale bar, 12 µm. (B) 4T1 tumor cell growth was visualized at day 1, 3 and 7 following tumor implantation through the dorsal skinfold chamber. A HIF-1 reporter (HRE-GFP) was expressed in tumor cells (CMV-RFP) and both were visualized using fluorescence microscopy. The vasculature is shown in red and was visualized using white light. Scale bar, 300 µm. Reprinted from¹⁰² with permission from Elsevier.

Figure 2. Most frequently used imaging windows in cancer research. (A) The CIW: the coverslip is glued onto the skull. The image depicts a coverslip and a metal ring. (B) The DSC: the metal frame of the window is clamped around the dorsal skinfold. Photograph shows the window chamber, retaining nuts and glass window. Scale bar, 1 cm. Photograph reprinted from³⁹ by permission from Macmillan Publishers Ltd: Nature Protocols. (C) The MIW: a purse-string suture tightens the skin around the MIW. The photograph depicts the titanium MIW. . (D) The AIW: a purse-string suture secures the AIW onto the abdominal wall and skin. The photograph depicts the titanium AIW with the groove in the side and is adapted from.³² All cartoons from³² Reprinted with permission from AAAS.

Table 1. Window surgery equipment

Equipment	Company	Product	Window
Anesthetics scavenger	VET tech solutions LTD OR Harvard Apparatus	Active scavenger unit Fluovac	GEN GEN
Antiseptic surgical scrub	Betadine	Betasept Antiseptic Surgical Scrub	GEN
Balance	Denver Instruments	Multiple available	DSC
Buprenorphine hydrochloride	BD pharmaceuticals limited	Temgesic (0.3 mg/ml)	GEN
Clipper	Wella	Contura clipper	GEN
Cover glass	Warner Instruments VWK international Thermo Scientific	Small Diameter Coverglass (5mm, #1) Coverslips round 6 mm Menzel Glazer 12 mm	DSC CIW MIW, AIW
Cyanoacrylate glue	Henkel	Pattex Ultra Gel	CIW, AIW
Dental cement	Vertex dental	Vertex Self-Curing	CIW
Drill	Dremel	Dremel 3000 (dental drill may be more convenient) + 105 Engraving Cutter	CIW
Opthalmic ointment	Kela Veterinaria	Duodrops veter kela 10 ml	GEN
Forceps	Roboz surgical instruments Co Neolab-DUMONT	Graefe forceps curved, serrated Forceps No. 5, smooth tying platform	GEN GEN
Heating pad	Inventum Holland VB		GEN
Hypnorm	VetaPharma Ltd	Hypnorm (Fentanyl citrate 0,315mg/ml+ Fluanison 10mg/ml)	CIW
Induction cage	Techniplast	Induction cage with autoclavable filter top	GEN
Inhalation anesthetic	Abbott OR Baxter	Isoflurane (100% w/w solution) Forane, Liquid for inhalation	GEN
Isoflurane Vaporizer	Isotec OR Vet Tech solutions LTD	Anesthetics machine	GEN
Midazolam	Actavis	Midazolam Actavis 5mg/ml	CIW
Needle	BD microlance	16 gauge needle	DSC
Needle holder	Aesculap		GEN
Scissors	Roboz surgical instruments Co	Microdissection scissors 3.5 inch	GEN
Silicone oil	Sigma- Aldrich	Silicone oil	CIW
Stereotaxic frame	Stoelting	Lab standard™ stereotaxic, rat and mouse	CIW
Sterile surgical gloves	SemperMed		GEN
Sterilizer	Cell point Scientific	Germinator 500	GEN
Surgery light	Schott	Ace I	DSC
Surgical drape	Medline industries		GEN
Surgical stereo microscope	Olympus		DSC, CIW
Sutures	Ethilon/Ethicon	Polypropylene sutures (4.0)	DSC, MIW, AIW
Vital sign monitors	Starr Life Sciences Corp.	MouseOx Plus	GEN
Window (chamber) frame	FMI instrumed AJP Trading Co. INC	Titanium AIW or MIW: STMF136–02A that meets ISO5832 part 3 and EN10204–3.1.B Window chamber, C-holder, screws, retaining ring	MIW, AIW DSC

The window column indicates for which type of surgery the equipment is used: DSC, CIW, MIW, AIW or GEN (general equipment for all surgeries).

Table 2. Troubleshooting imaging window surgeries

Problem	Possible reason	Solution DSC	Solution MIW	Solution AIW	Solution CIW
Mouse dies during surgery	Hypo/Hyperthermia	Control the power of the heating pad and measure mouse temperature.			Control the power of the heating pad and measure mouse temperature.
	Excessive anesthesia	Reduce the amount of anesthesia.			Adjust the amount of anesthesia per mouse weight.
Bleeding during drilling and dura mater removal	Vascular damage	NA			Control the bleeding by the Gelfoam sponge. Precisely perform these procedures under a stereomicroscope.
The window falls out	The suture was placed too close to the skin edge.	NA	Mouse needs to be sacrificed because the tissue underneath the window is most likely dehydrated.
	The dental cement was applied on the skin				Glue the skin and apply the dental cement on the skull.
Bleeding underneath the coverslip	Vascular damage	Be more careful during surgery; cauterize wound.	Remove the liquid under the window as indicated above (Table 4). Be more careful during surgery; cauterize wound.
The mouse head moves	The mouse head is not properly fixed on the stereotactic frame.	NA			Ensure that the mouse head is firmly fixed by ear bars and a teeth holder.
The abdominal organ moves underneath the window	Too little glue was used to fix the organ to the window.	NA		Wait a day. Sometimes organ settling takes about 24 h.	NA
				Use gels like CyGel to reduce organ movement.^12
			Take out the window by cutting the stitch and place it again.
There is an infection underneath the window (white cloudy liquid)	No aseptic working environment was maintained or the instruments were not sterile.	Euthanize the mouse. Next time provide antibiotics.

NA indicates not applicable.

Table 3. Clinical parameters of distress

Parameter	Signs	Cause
Activity level	Hypo activity (hunched, lethargic), hyperactivity, restlessness, lack of curiosity.	Pain.
Attitude	Depression, no awareness of surroundings.	Depression, pain.
Behavior, spontaneous	Squealing, self-trauma, isolation from cage mates.	Pain.
Behavior, Provoked	Vocalization, teeth grinding, struggling, hiding, aggressiveness, unwilling to move, guarding the incision site.	Pain.
Body Condition	Tenting of the skin, missing anatomy, hydration, anorexia, absence of feces and urine.	Reduced food and fluid intake.
Fur and skin	Ruffled or greasy or dull fur, porphyrin (red) staining around eyes and nose, cyanotic or pale ears, feet, tail, skin lesions, soiled anal or genital area. Redness and swelling at incision site.	Failure to groom. Excessive licking/scratching. Pain. Infection.
Extremities	Cold, blue, hot or red.	Hypo or hyperthermia.
Eyes	Clarity/condition of lens, sunken or protruding eyes.	Pain. Failure to groom.
Posture	Hunched back, tucked abdomen, head tucked down.	Pain.
Locomotion	Lameness, favoring a limb, upright or downwards.	Pain, physical blockade.
Neurological	Tremor, convulsion, weakness, circling, paralysis, head tilt, coma.	Neurological trauma.
Vital Signs	Respiratory distress (open mouth breathing, agonal breathing, pronounced chest movement).	Shortage of oxygen. Pain.

Figure 3. CIW surgery. (A) The mouse head is shaved and fixed on a stereotaxic frame. (B) The skin is cut in a circular manner around the skull and the periosteum is scraped. (C) 5mm diameter circle is drawn around the region of interest. (D) A groove is drilled around the region of interest. (E) Cold cortex buffer is applied during drilling. (F) The craniotomy is lifted under a drop of saline exposing the brain. (G) The brain is covered with silicone oil and sealed with a 6 mm diameter coverslip. (H) Dental cement is applied to cover exposed skull and a stainless steel ring is glued parallel to the coverslip. (I) The mouse is injected with 100 μg/kg of buprenorphine and allowed to recover on a heating pad.

Table 4. Troubleshooting imaging

Problem	Possible reason	Solution DSC	Solution MIW	Solution AIW	Solution CIW
Poor visibility	Liquid accumulation or “membrane” formation can interfere with imaging.	Remove the O-ring and the coverslip to get access to the tissue. Use forceps or a cotton tip applicator to remove the membrane or liquid. Then replace the coverslip and O-ring.	Remove the liquid under the window by inserting a 25-G needle and slowly filling the syringe. Avoid hitting the organ or breaking the coverslip.		NA
	The tissue of interest is too far from the objective.	NA	If using an inverted microscope, wait for 30–45 min until the tissue gets closer to the objective by gravity.
		NA			Reduce the amount of silicone oil between the coverslip and the brain. The coverslip should be glued at the minimal distance from the brain.
The image is dark	When water objectives are used, the amount of water in between the objective and coverslip may be inadequate.	Supply water to the objective manually or use a dispenser that can regulate water supply automatically.			The use of an oil-immersion lens or a dry objective can be considered as well. However, often a trade-off must be made with a reduction in NA.
Bleeding under the coverslip	Vascular damage	Remove the liquid under the window as indicated above.			If tumor is still visible take no action.
					If extensive bleeding is obstructing tumor visualization, bring mouse under anesthesia and break the window, remove the blood and place a new window.
Mouse dies during imaging	Sedation is too deep.	Establish appropriate sedation level for your experiment. For long-term experiments a low amount of isoflurane is recommended. Control mouse breathing and maintain the temperature at 36ᵒC. If breathing is irregular decrease the amount of isoflurane.
	Mouse is hypo/hyperthermic and/or dehydrated	To avoid dehydration inject the animal with saline prior or during the imaging session. Maintain the temperature of the mouse at 36C using a heated blanket or a heated climate chamber that surrounds the microscope.
Mouse/organ moves during the imaging	Sedation is inadequate. The mouse is not fully unconscious or is breathing irregularly.	NA	Establish appropriate sedation for your experiment.
			Firmly fix the IW to the box with tape to avoid peristaltic movement or breathing artifacts.
			Make use of a resonant scanner to increase the speed of image acquisition.
			Use advanced motion compensation methods to reduce movements.^103^,^104
Tissue movement or deformation during imaging	Tissue is compressed if the mouse is imaged in a non-anatomical position. Eg. Brain imaged through a CIW on an inverted microscope	NA			Use image-processing software to correct the image deformation.

NA indicates not applicable.

Figure 4. Purse-string suture fixes the AIW into the animal. (A) A purse-string suture is placed. The suture is first placed through skin and then through opposing abdominal wall. The outer loops (OL) are not tightened, whereas the inner loops (IL) are. (B) Once the purse-string suture is placed it should contain 4 or 5 outer loops. (C) The AIW is glued to the organ (liver [L]) and the skin and abdominal wall are placed within the window groove. (D) The sutures are tightened to secure the AIW in place. All pictures adapted from³¹ by permission from Macmillan Publishers Ltd: Nature Protocols.

Figure 5. Fixation of the window during imaging. (A) A custom-designed imaging box that can be mounted on top of the stage of an inverted microscope. Reprinted from³¹ by permission from Macmillan Publishers Ltd: Nature Protocols. (B) The imaging box shown from below. MIW is fixed in the center hole. (C) Inserts for the imaging box. A CIW window insert and an AIW insert are shown. (E) An imaging mount that can be used to fix a DSC onto a microscope stage. The three smaller holes allow the bolts on the window chamber to pass through and fix it, and the large hole permits imaging through the window. Scale bar, 1 cm. reprinted from³⁹ by permission from Mark W. Dewhirst and Macmillan Publishers Ltd: Nature Protocols. (F) An inverted Leica SP5 microscope with a dark climate chamber surrounding the stage is shown. Reprinted from³¹ by permission from Macmillan Publishers Ltd: Nature Protocols.

Figure 6. Retracing of the exact same field of view over multiple days. (A) A microscope with a motorized stage allows storage of regions of interest that can be imported at request. Reprinted from¹² by permission from Macmillan Publishers Ltd: Nature. (B) The numbered grids on a gridded cover glass can be visualized using reflection microscopy. The numbers can be used to retrace the same position over multiple days. Reprinted from³¹ by permission from Macmillan Publishers Ltd: Nature. (C) The vasculature can be used as reference point to retrace a field of view. The microvasculature (green) is imaged through a cranial imaging window. Intravascular proliferation of injected PC14-PE6 lung carcinoma cells (red) can be seen from day 3 onwards. Images reprinted from²² by permission from Macmillan Publishers Ltd: Nature Medicine. (D) In the liver the type I collagen network (purple) visualized by second harmonic generation imaging was used to retrace the same position of multiple days. To show retracing, the images of day 1 and day 2 were given a false color (red or green respectively) and were merged. Yellow indicates colocalization. To quantify the amount of colocalization, the pixel intensity values of day 1 and day 2 in the merged image were displayed in a scatter plot. The Pearson correlation coefficient (r) was calculated and the non-colocalizing pixels were displayed in falce colors (red and blue) on top of the day 1 image displayed in gray. Scale bar, 20 µm. Image from³² Reprinted with permission from AAAS. (E) Dendra2 labeled tumor cells (green are non-photoswitched cells and red are photoswitched cells) imaged through a mammary imaging window. Images were taken 0, 6 and 24 h after photoswitching. Scale bar, 30 µm. Images reprinted from by²⁹ permission from Macmillan Publishers Ltd: Nature Methods.

Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell 2004; 5:429 - 41; http://dx.doi.org/10.1016/S1535-6108(04)00115-1; PMID: 15144951

 PubMed Web of Science ®Google Scholar

Palmer GM, Fontanella AN, Shan S, Hanna G, Zhang G, Fraser CL, Dewhirst MW. In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters. Nat Protoc 2011; 6:1355 - 66; http://dx.doi.org/10.1038/nprot.2011.349; PMID: 21886101

 PubMed Web of Science ®Google Scholar

Ritsma L, Steller EJ, Beerling E, Loomans CJ, Zomer A, Gerlach C, Vrisekoop N, Seinstra D, van Gurp L, Schäfer R, et al. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci Transl Med 2012; 4:ra145; http://dx.doi.org/10.1126/scitranslmed.3004394; PMID: 23115354

 PubMed Web of Science ®Google Scholar

Ritsma L, Ellenbroek SI, Zomer A, Snippert HJ, de Sauvage FJ, Simons BD, Clevers H, van Rheenen J. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 2014; 507:362 - 5; http://dx.doi.org/10.1038/nature12972; PMID: 24531760

 PubMed Web of Science ®Google Scholar

Vinegoni C, Lee S, Feruglio PF, Weissleder R. Advanced Motion Compensation Methods for Intravital Optical Microscopy. IEEE J Sel Top Quantum Electron 2014; 20; http://dx.doi.org/10.1109/JSTQE.2013.2279314; PMID: 24273405

 PubMed Web of Science ®Google Scholar

Lee S, Vinegoni C, Sebas M, Weissleder R. Automated motion artifact removal for intravital microscopy, without a priori information. Sci Rep 2014; 4:4507; http://dx.doi.org/10.1038/srep04507; PMID: 24676021

 PubMed Web of Science ®Google Scholar

Ritsma L, Steller EJ, Ellenbroek SI, Kranenburg O, Borel Rinkes IH, van Rheenen J. Surgical implantation of an abdominal imaging window for intravital microscopy. Nat Protoc 2013; 8:583 - 94; http://dx.doi.org/10.1038/nprot.2013.026; PMID: 23429719

 PubMed Web of Science ®Google Scholar

Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, Winkler F. Real-time imaging reveals the single steps of brain metastasis formation. Nat Med 2010; 16:116 - 22; http://dx.doi.org/10.1038/nm.2072; PMID: 20023634

 PubMed Web of Science ®Google Scholar

Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE, van Rheenen J. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 2008; 5:1019 - 21; http://dx.doi.org/10.1038/nmeth.1269; PMID: 18997781

 PubMed Web of Science ®Google Scholar