Essay: In Vivo Fluorescence Microscopy: Surgical and Therapeutic Uses

Today in the world of medicine various types in vivo fluorescence microscopy is being used to guide and innovate various surgeries and therapies. There are several types of in vivo fluorescence microscopy (IVFM), for example confocal and 2-photon microscopy. By the use of these techniques it is possible to image very small microstructures, in a living organism without the risk of serious harm to the organism or the parts of it that are being examined. Some of the materials involved in these microscopies are fluorescent proteins such as the green fluorescent particle (GFP), red fluorescent protein (DsRed), and any other of the various color-proteins that have been derived from the original GFP like cyan fluorescent protein or yellow fluorescent protein. This method of imaging shows quite a bit of promise in terms of applications and uses in surgeries and therapies, and can be used to vastly improve the accuracy of various techniques.
These microscopies are very useful, but in order to use a microscopy one obviously then would need a microscope capable of achieving it. As for the confocal fluorescent microscopy a laser provides the highly excited light used in the microscopy. The laser is reflected off a dichroic mirror, the laser then bounces off two mirrors which are mounted on a motorized device that is then used to scan the desired tissues. The light bombards the selected fluorophores which then emit the light needed at a particular wavelength and is ‘descanned’ by the same dichroic mirrors, this emitted light passes through the mirrors because they only reflect certain wavelengths but allow other ones to pass through them. The light that passes through is focused onto a pinhole and the light passing through it is measured by a detector and is interpreted by a computer to generate the image. This setup is then used in-tandem with some existing therapy or surgery to improve accuracy of the treatment. Similar to confocal microscopy, two-photon microscopy involves taking a high powered laser capable of bursts of infrared light at a very high rate (measured in femtoseconds) is used in tandem with fluorescent molecules, this light is then reflected by a dichroic mirror to the specimen and focused within it. The volume of what is being examined is then raster scanned by a galvanometer-driven scanner in addition to a piezoelectric objective in order to image the specimen in three dimensions. The light emitted is collected by the same objective bounced back onto the dichroic mirror along the path of emission and this light is gathered and composed into an image. An addition is used to help filter out scattered excited light, and a single-photon detecting device is placed behind the dichroic mirror and is used to help determine the maximal efficiency of the configuration of the 2-photon setup so as to determine when adjustments need to be made in order to get clearer imaging. This is how the two microscopies are setup, and how they work.
Now, as for how these amazing microscopies are being applied in the world of medicine to improve and innovate surgeries and therapies of all kinds, there are dozens of examples of the numerous benefits that these microscopies have offer. For example:
1. Mesenchymal Stem Cell (MSC) Delivery – For the administration of stem-cells it is imperative to deliver the cells to the selected tissue with precision and in very precise amounts. One way scientists have been using IVFM to improve an existing therapy comes to us by way of confocal microscopy’by employing this technique one can far more easily quantify the amount of cells being administered, as well as improve the precision of locally delivering the cells. Thereby greatly increase the therapeutic potential of certain cell-based therapies of this kind. In addition, this allows us to reduce the amount of cells being used down to just what is needed for the therapy thus lowering the amount of material gone to waste.
2. Blood-vessel closure using photosensitizers engineered for two-photon excitation – An example of in vivo fluorescence microscopy being used as a therapy is the use of 2-photon microscopy (or specifically the excitation of the photons that is provides) is being used in photodynamic therapies, wherein the 2-photon excitation caused by the fluorescence microscopy is used in tandem with photosensitizer drugs to activate the drugs absorbed by the tissue, and thanks being to the precision of the 2-photon excitation we are able to control the amounts of drug active in the targeted tissue with extreme precision considerably improving certain therapeutic methods. For example, by using photosensitizer drugs specifically engineered porphyrin dimers that have cross-sections designed to respond efficiently to 2-photon excitation we have used the technique of 2-photon to selectively manipulate and close blood vessels by activating the dimers very precisely where we want to achieve the desired effect.
3. Brain Surgery ‘ Confocal: Detecting and analyzing brain tumors during the actual surgery is important to ensure precise excision and to determine that all of the tumor has been successfully removed while, at the same time, making sure that there is as little undue cutting of the brain as possible. Most of this detection and evaluation is ex vivo, that is the tumorous parts of the brain are removed and biopsied to analyze them, however this takes some time, and time is not a luxury afforded to a patient under the knife. Surgeons then make use of a fluorescent protein or dye (which one that is selected is determined beforehand, and there are many to choose from; the best one is decided upon and used to achieve fluorescence), if selected correctly the dye or protein should be more readily absorbed by the tumors than the surrounding noncancerous tissue, and then by using a confocal fluorescent microscope it is possible to very quickly ‘see’ the cancer without actually having to remove part of the brain for a lengthy and potentially unfruitful biopsy. This improves the accuracy of resection, and at the same time lowering the chance of serious brain damage in the patient, and lowering the chance of infection due to drastically lowering the amount of time needed to perform the surgery. A single optical fiber is used to direct the light for excitation and it is also the ‘pinhole’ needed to filter out scattered light and achieve an accurate image using confocal microscopy. Also a solid-state laser is used at a wavelength of 488 nm with the emission being detected in the range of 505-585 nm, and this method allows a view as deep down 250 ??m, and the optical slice imaged is about 7 ??m in thickness.
4. Breast Cancer Surgery ‘ Confocal: Typically in breast surgery the tumorous tissue is examined using core needle biopsy. The tissue is removed and either frozen or treated with formalin and then embedded in a paraffin wax to be examined. Freezing the tissue can cause cellular death or damage and the paraffin wax has a high risk of artifacts impeding assessment, and both processes take a decent amount of time to perform. By using confocal fluorescence microscopy and an optical contrast agent such as proflavine dye it was possible for surgeons to examine the biopsied material with higher resolution and in a much shorter span of time simply by dying and looking at the tissue with a confocal fluorescence microscope, and due to the ease of this technique they were also able to use it in vivo to examine and assess the same cultures that they previously could only see accurately ex vivo. Needle core biopsy is not particular invasive but the use of confocal fluorescence microscopy is a couple of steps ahead of the previous method and is does not have to be invasive whatsoever. For this technique a laser with an excitation wavelength of 488nm and a 550/88nm bandpass filter, 750×750??m images are taken and then stitched together to form a composite image and then used to evaluate the tissue.
5. Intracardiac Cell Transplant Screening ‘ Two-Photon: Transplanting and grafting heart muscle cells onto a heart in order to improve the contractile strength of the organ has been shown to be an effective way to improve heart function. In order for the grafted cells to contribute to the contractions of the heart the individual cells have to form working syncytium with the health host cells of the heart’s contractile muscles. By using two-photon fluorescence microscopy it is possible to examine the cells in situ/in vivo and determine how well the grafted cells are taking. For the procedure a high-intensity laser projects a laser with a wavelength greater than 700nm is used to excite the chosen fluorphore and the excited wavelength emitted is 850nm. Using this technique increases the over effectiveness of the therapy greatly and allows us to deliver the cells in a way that better ensures that they take or at least determine where they will not so as to conserve the available cellular materials.
Conclusion:
Confocal and two-photon fluorescence microscopy have had a great impact on the existing surgeries and therapies in medicine. They are both involve the same basic tools to achieve imaging, although they do so differently. Having both of these techniques at our disposal is invaluable because even though two-photon microscopy provides higher resolution images there are times where confocal is more practical to use than two-photon such as when the exposure of the high frequency light used to image with the two-photon technique causes phototoxicity in the specimen, bearing in mind that these methods are being used in vivo, it is imperative that cellular death does not occur as a result of the treatment, and it is here that the confocal finds its use. Likewise there are times when confocal is not possible due to pigmentation in the tissues being examined that cause so much light to be scattered that confocal is totally useless; the high frequency infrared light of two-photon exceeds confocal in this regard because due to the very nature of it, it has a stronger capability to penetrate into tissue and not be diffused or scattered allowing for clear imaging. It is the combination of these techniques and their application to surgeries and therapies that make them such great innovations. They allow for much faster, and higher resolution imaging that previous conventional methods could not achieve and most importantly they may be used in a living organism, and many older practices required that the specimen be excised or dead in order to get an image. These types of imaging are a vast improvement over what we had, and the implications for surgeries and therapies are amazing and numerous.