The importance of in vivo measurements became evident already in 1839, when intravital imaging was first described by Wagner. He observed the interaction of leukocytes with the blood vessel wall in the webbed feet of a grass frog using bright field transillumination.5 In these early days, most intravital imaging studies could only examine the vasculature and the microcirculation, due to the old-fashioned optics and the lack of contrasting techniques to visualize other tissues. This changed in 1911, when Heimstadt introduced the first fluorescence microscope.6 Together with the development of exogenous fluorophores,7 the fluorescence microscope allowed in vivo imaging of intravenously injected fluorophores and these “circulating sources of light” were visualized in several tissues such as liver and kidney.8 Nonetheless, the earliest observations were done merely by eye or diafilms,9 limiting these studies to descriptive reports on relatively large-scale events.
In the mid- and late 20th century new lasers, optics and detectors were developed, leading to the introduction of video (widefield) and confocal microscopy. In contrast to earlier versions of fluorescence microscopes, these advanced microscopes could record images and therefore allowed post-acquisitional image analysis and publication of the experimental images.10,11 In addition, the confocal microscope was designed to eliminate out-of-focus emission light (from beyond the optical section) by virtue of a pinhole, resulting in contrast enhancement (making subcellular structures clearer) and improved Z-resolution. Thereby it constructs optical sections through tissue without physically sectioning it (Fig. 2A). Since its first biological implementation in 1987,12,13 confocal microscopes have been applied to study numerous (subcellular) events in living mice such as tumor cell migration in mammary tumors.14 Spinning disk confocal microscopes have been used in several studies because it allows fast scanning and therefore induces little phototoxicity.15
Figure 2. Single-photon microscopy versus two-photon microscopy. (A) A Z-stack of images of a mouse mammary tumor (KeP1_11 Dendra2) was acquired with single-photon (1P, confocal microscope) and two-photon (2P, two-photon microscope) excitation in a living mouse. The imaging depth is indicated in every image. Note that deep inside tissue good contrast can only be obtained when using a two-photon microscope. (B) Jablonski diagram showing single-photon and two-photon absorption and emission by a fluorophore. For second harmonic generation (SHG) imaging excitation does not take place; instead, two photons scatter simultaneously, thereby generating a single photon in the visible light range. Lower image; two focal points were bleached in a fluorescent plastic using single- (left) and two-photon (right) excitation. Bleaching profiles are shown in XZ. Note that single-photon excitation creates a cone-like structure, while two-photon excitation is restricted to the focal point. (C) Intravital images of the same region in which scatter- or SHG-signal from ECM fibers was acquired. Scalebar represents 20 μm.
Nowadays, the most advanced confocal microscopes are equipped with several lasers in the visible light spectrum (400-650 nm) to excite a diversity of fluorophores. However, scattering of visible light in tissue limits the imaging depth. This problem is overcome by exciting fluorophores with infrared lasers (<1000 μm deep)16 (Fig. 2A). In 1990, Denk et al. developed the ultrashort pulsed laser17 in which all photons over a time span of 12.5 ns are concentrated into a femtosecond pulse (for a 80 MHz pulsed laser). In the focal plane, the photon density within such a pulse is sufficient to make fluorophores absorb two photons nearly simultaneously (Fig. 2B) (this was predicted already in 193118 and validated in 1961).19 This technique, often referred to as two-photon microscopy, renders elimination of out-of-focus light by a pinhole redundant, since the excitatory photon density and thereby the two-photon excitation effect is strictly confined to the confocal plane (Fig. 2B). This simplifies the optical path, because emitted light does not have to travel back through the scanner with losses of light at the pinhole and mirrors, but instead can be detected close to the objective.20 The advantages of two-photon microscopy result in low bleaching and minimal phototoxicity deep inside living tissue.
To visualize individual cells within various organs of the mouse, the imaging depth of two-photon microscopy is not sufficient and requires surgical exposure of the imaging site.21 However, these surgical procedures limit the duration of imaging sessions to several hours. Long-term intravital imaging (up to 30 hrs) of surgically exposed tumors in mice has been achieved by optimized imaging conditions (e.g. monitoring vitals).15,21 However, by using the dorsal skinfold chamber, Lehr and coworkers pioneered high-resolution intravital imaging over multiple days.22 At the present time, this chamber is widely used for multi-day imaging of the vasculature and tumors (e.g.23). More recently, other types of chambers have been developed by us and others to study mammary tissue,24,25 brain tissue,26 and the spinal cord.27 Although imaging windows for the lung have been designed, they have not yet allowed imaging for more than a few hours.28
To monitor individual cells over multiple imaging sessions, vascular and extracellular matrix structures and tattooed reference marks have been used as roadmaps in healthy tissue in order to repeatedly trace back studied cells.29-31 In this manner, formation of metastasis has been followed for weeks (LR and JvR, unpublished) with the animals well recovering from anesthesia in between the imaging sessions. If, however, tissue topology changes over time (e.g. in tumorigenic tissue), areas of interest can be retraced by taking advantage of photoconvertable fluorophores such as Dendra232 and PSmOrange.33 For example, violet illumination shifts the absorption and emission spectra of Dendra2 towards longer wavelengths converting green Dendra2 to red Dendra2 fluorophores. We have used Dendra2 to photo-mark and track individual cells and groups of cells for multiple days34 (Fig. 3B) and could, thereby, visualize distinct migratory behavior of tumor cells. For example, we showed that tumor cells surrounding large blood vessels are more migratory than tumor cells surrounding capillaries.24 This assay has high potential to test the effects of clinically relevant pharmacological agents in vivo as was done for agents that target integrin–Fak–Src signaling.35 Thus, the combination of photoconvertable fluorophores and imaging windows provides a powerful tool for long-term intravital imaging at high resolution.
Figure 3. Visualizing cell migration over long and short periods of time. Intravital imaging of mouse tumor cells was performed using two-photon microscopy. (A) Stills of a time-series showing KeP1_11-Dendra2 cells migrating along type I collagen fibers. (B) C26-Dendra2 cells were imaged through a mammary imaging window after photomarking a subpopulation in a square region (dashed square) by violet light-induced photoconversion of Dendra2. Twenty-four hours later we rescanned the region. Note that the converted cell population has relocated (see dashed line). The scale bars represent 20 μm.
Taken together, we are now able to detect fluorescence at high resolution, deep inside living tissue and over multiple days. Therefore, it comes as no surprise that intravital imaging is becoming increasingly popular to study the behavior of fluorescently labeled cells in the in vivo setting and, as will be discussed below, signaling events between and within these cells.
In order to study signaling events between cells, multiple cell and/or tissue types must be visualized simultaneously. Contrast agents are often used to distinguish between different cell types and tissues. However, certain substances (such as ECM, blood vessel structures) are detectable without labeling. For example, optical frequency domain imaging (OFDI) detects the differential optical scattering properties of various tissue structures such as blood and lymphatic vessels and has (already) been used to study angiogenesis and lymphangiogenesis.36 As another example, reflectance imaging (also referred to as back-scattering) is based on the detection of excitation light that is scattered by solid-state structures resulting in contrast between dense and less dense tissue.37 Reflection of the excitation light occurs at the interface between structures with differential refractive indices such as ECM components and water (Fig. 2C). Although the signal is not specific, reflectance imaging can visualize many microenvironmental structures ranging from ECM or blood vessels to stromal cells (e.g. see refs. 37 and 38). A more specific and regularly used technique to visualize collagen fibers is second harmonic generation (SHG) microscopy (Fig. 2C). At highly ordered and non-centrosymmetric structures, such as Type I collagen, the energies of two photons are combined and reflected as a single photon with doubled energy and half the wavelength. Typical wavelengths used for SGH-imaging range between 800 and 1300 nm, since the SHG-signal can then be detected using standard fluorescence microscopy filter sets. Since SHG and reflectance imaging are not based on fluorescence phenomena, they do not excite molecules and therefore do not induce photobleaching. These techniques can be used to study Type I collagen in various healthy and pathogenic tissues including skin, gut and breast (e.g. ref. 39). For example, SHG has proven to be highly usefull when studying cancer cells; it has been shown that breast cancer cells migrate along collagen-I fibers with high velocity and persistence (Fig. 3A).1
Another way to visualize cells and tissues without labeling them is by detecting endogenous fluorescent molecules which are present in almost every cell type such as tryptophan, pyridinic and flavin co-enzymes; their emission is commonly referred to as autofluorescence (see e.g. ref. 40). Since both excitation and emission spectra of these components have considerable overlap, the fluorescence level cannot be used to distinguish among cell types. However, the fluorescence lifetime (the average time an excited fluorophore resides in its excited state) does differ among the various components (e.g. tau-NADH ~ 1 ns and tau-FAD ~ 3 ns)41 and can thus be measured using FLIM (fluorescence lifetime imaging microscopy). The different molar ratios of these autofluorescent components, as present in distinct cell-types and tissues, will constitute cell type specific (average) autofluorescence lifetimes. Thus, imaging the (average) lifetime of auto-fluorescence has a strong potential for distinguishing multiple cell-types (Fig. 4A). This technique has already been successfully applied to discriminate healthy from tumor tissue42-44 and to predict tumor grade parameters, such as metastatic potential.45
Figure 4. FLIM and FRET measurements in a living mouse. (A) Discriminative visualization of multiple tissue components by Fluorescence Lifetime Imaging Microscopy (FLIM). Shown are the autofluorescence and lifetime image. Note that blood vessel (e.g. asterisk) and cells (e.g. arrow) have distinct average lifetimes. (B) Cartoon of the N-Wasp FRET biosensor. In its inactive form, the CFP- and YFP-moieties are in close proximity so that FRET can occur upon CFP excitation, resulting in YFP (~540 nm) emission. When the N-Wasp sensor core is activated, the sensor will unfold, leading to a loss of energy transfer. This is reflected as increased CFP and decreased YFP emission. (C) Ratiometric FRET imaging performed on MTLn3 mammary tumor expressing the N-Wasp FRET biosensor. CFP excitation induces CFP (left panel) and YFP emission (middle panel); the FRET ratio image (CFP/YFP; right panel) reflects N-Wasp activity. Note the small membrane protrusion in which N-Wasp is active. Scale bar represents 10 μm.
As mentioned above, cell types and tissues can also be distinguished by using contrast agents. However, most contrast agents dilute quickly, for example due to cell division. When short term labeling is adequate, blood vessels can be visualized by angiography, i.e. injection of fluorescently labeled high-molecular weight dextran or quantum dots into the blood stream. As another example, intravenously injected low molecular weight-dextran molecules (<70 kD) leak out of the blood into the surrounding tissue, where they are taken up by phagocytic immune cells such as macrophages. Moreover, dendritic cells can be labeled by intracutaneous injection of carboxy-fluorescein diacetate succinimidyl ester (CSFE).46 Immune cells can also be specifically labeled by transferring isolated cells into recipient mice after labeling them ex vivo with tracker dyes.47 A general drawback of the latter approach is that these labeled cells will only constitute a small fraction of the total blood cell population of the recipient animal. Alternatively, cell-type specific fluorescent antibodies (e.g. for lymphatics, see ref. 48; for Gr1+ myeloid cells, see ref. 15) can be injected to establish contrast between different cell types. Especially for immune cells that can be adoptively transferred, these approaches enable researchers to label differential cell types with specific colors and visualize their physical interactions in vivo (e.g. ref. 49).
Since the introduction of genetically encoded fluorophores such as green fluorescent protein (GFP), it has become possible to label non-hematopoietic lineages that could not be adoptively transferred, such as cancer cells with an epithelial origin. For this, fluorescent protein (FP) expression, driven by cell-type specific promoters, has been the preferred method to visualize cell types in intravital imaging experiments. Advantages of these FPs are that they (1) are non toxic, (2) are not limited in tissue penetration (as labeled antibodies or dyes are) and (3) come into place non-invasively. Thanks to a variety of FP color variants, multiple cell types can currently be genetically labeled and simultaneously visualized (e.g. blue, green and red fluorophores). This has led to the exciting possibility to study interactions and communication between different cell types in real time. For example, Egeblad and coworkers visualized the behavior of different stromal cells in mammary tumors and showed that most stromal cells exhibit higher motility at the tumor edge than when residing within the tumor mass.15 In another study, Wyckoff and colleagues visualized how signaling between mammary tumor cells and macrophages drive metastasis.50 By co-registration of GFP-expressing mammary tumor cells and Texas Red-dextran labeled macrophages they showed how macrophages assist the tumor cells in invading the stroma and entering the blood stream.50 Macrophages were also found to be involved in the development of mycobacterial liver granulomas.51 In yet another study it was shown that T cell interactions with dendritic cells are governed by T cell sensing of the antigen dose, creating a threshold for its activation.52 Nowadays, many new fluorescent proteins with additional properties have been designed, opening new avenues for intravital imaging of cell-cell signaling. For example, fluorescent proteins like LSS-mKate153 have been designed for visualization in combination with CFP and GFP, since e.g. mKeima combines a CFP-like absorption spectrum with a mRFP-like emission spectrum. Piatkevich and co-workers used this multiple-channel approach to visualize that tumor cells are more polarized when they are in close proximity with blood vessels than when they are located far away from the circulation.53 Finally, far-red fluorescent proteins such as mNeptune, eqFP650/670 and iRFP have been developed to allow imaging deep inside tissue, thanks to their excitation and emission spectrum that are near-infrared.54-56 For a good review on the latest developments of fluorescent proteins see ref.57
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