Historically, significant milestones in the evolution of life science and technology have led to advancements in the development of microscopy techniques. From visualization of microbial cells to studying subcellular components, tissues, and dynamic physiological processes, imaging techniques have advanced at every level. The introduction of fluorescence and confocal imaging techniques has allowed the visualization of two-dimensional and three-dimensional structures, respectively, with high sensitivity and improved resolution. However, these techniques could not penetrate deeply into the tissues and produce clear images.
Over time, the limitations of the single photon linear techniques for microscopy have been resolved with the discovery of nonlinear, laser scanning, two-photon excitation microscopy, also known as Multiphoton microscopy. In 1931, Göppert-Mayer, a German-American physicist described the theoretical concept of multiphoton excitation, during the early development of quantum optics. Using this concept and redshift light, it was experimentally demonstrated for the first time as the multiphoton microscope. An alternative to the confocal and deconvolution technique, multiphoton microscopy is based on the simultaneous absorption of two-photon by the fluorophore for the occurrence of excitation and emission.
Today, it is considered a technique of choice for imaging thick tissues, and subcellular components at the molecular level, optical sectioning, examining cell functions, and studying biological interactions with less invasion for longer duration, minimal phototoxicity, and photodamage. This technique of microscopy is widely used in biological research and medical science including neuroscience, immunology, endoscopy, cancer research, genomics, and engineering.
Principle of Multiphoton Microscopy
The fluorophore is transitioned to the state of excitation electronically with the simultaneous absorption of two photons and a combination of their energies at higher photon densities. It is known that energy is indirectly proportional to the wavelength of a photon. Therefore, the wavelengths of two photons should double those needed for single-photon excitation. This distinctive application of longer wavelengths of photons reaching the infrared spectrum excites chromophores in a single quantum event. In turn, they emit secondary radiation at shorter wavelengths.
The excitation of multiple photons depends on a rate constant proportional to the square of the intensity of excitation. In multiphoton fluorescence microscopy, a maximum level of fluorophore excitation is maintained by the high photon densities. In this regard, pulsed lasers with high power are used to accomplish the excitation level, ensuring no damage to the specimen. The laser emits short but intense pulses that increase the probability of the two photons absorption at a constant power level of the laser incident to a given fluorophore. For multiphoton fluorescence studies, common pulsed laser setups typically use short duty cycles of approximately 100 femtoseconds with repetition ranging from 80 to 100 MHz. This setup allows for effective image capture without exposing the sample to excessive heating and photodamage.
Instrumentation of Multiphoton Microscope
Although a multiphoton microscope is similar to a confocal microscope in terms of components, the use of ultrafast lasers in multiphoton microscopy makes it stand out. Its features such as high power and brief pulse duration excite the fluorophore. Moreover, this technique does not necessitate using a pinhole as required in confocal imaging instrumentation. The details of the components in a multiphoton microscope are briefly described below:
- Laser: The source of Infrared laser excitation emits a beam of laser to cause the absorption of photons in the sample. This is accomplished with the high peak power of irradiance and pulse duration of femtoseconds. Laser excitation sources such as Titanium (Ti) sapphire are preferred but costly, while ultrafast fiber lasers are newly used and less expensive, making this technique accessible and affordable.
- Mirrors: Mirrors with low group delay dispersion (GDD) are used in multiphoton microscopy. Its application reduces chromatic dispersion and random propagation of the pulse. These mirrors are equipped with dielectric coatings specifically designed to enhance reflectivity for the intended laser wavelength and reduce group delay dispersion (GDD). Dichroic mirrors with low GDD are utilized to differentiate emission signals at various fluorescence wavelengths, whereas low GDD scanning mirrors facilitate the transmission of emitted fluorescence to a detector.
- Filters: These include badpass, longpass, shortpass and dichroic filters. The excitation laser wavelengths are refined by bandpass or shortpass filters which also select specific wavelengths for detection and permit the passage of nonlinear signals. Often, dichroic filters are utilized to distinguish between excitation and emission wavelengths
- Objective: An objective directs the excitation source onto the sample, resulting in multiphoton absorption at the focal point. Through it, the resulting fluorescence is then assimilated back and transferred to the detector. Objectives designed for multiphoton microscopy generally possess a high numerical aperture (NA), high magnification, chromatic correction across a broad wavelength range, and a long working distance.
- Lens: The tube lens is used to focus the assimilated fluorescence and the image is viewed through it. It is situated at the back of the objective.
- Detector: A detector setup consists of a photomultiplier tube (PMT), imaging lens, and camera. It captures the fluorescence emitted from the focal plane, producing high-resolution images. Three-dimensional images can be created in the microscope by scanning through various focal planes and stitching the images together.
Sample Preparation in Multiphoton Microscope
The process of sample preparation in Multiphoton microscopy is essentially similar to those used in confocal or widefield fluorescence microscopy techniques. Multiphoton imaging can be performed on cell cultures, as well as on resected and labeled tissues, and even in vivo organs. In this technique, the presence of a noncentrosymmetric structure allows for the generation of the Second Harmonic Generation (SHG). In addition, Third Harmonic Generation (THG) can occur when there is a condition of negative phase mismatch, particularly in a non-homogeneous medium. These methods are highly significant in biomedical imaging primarily due to their ability to generate images without using labels.
Similarly, the selection of dyes for the sample preparation depends on the requirement of simultaneous excitation levels at different wavelengths. 4′,6-diamidino-2-phenylindole (DAPI) is considered unsuitable because it will interfere with all channels and if required, can be used for sequential imaging using the sequence manager. Furthermore, Alexa Fluor 488 dye is used when the laser is adjusted to 950 nm. Ultimately, users must choose dyes that can be simultaneously excited but have emission peaks that are sufficiently distinct for the filters to differentiate them into separate channels.
Operating Procedure of Multiphoton Microscope
The multiphoton microscope consists of a front and back system for imaging the sample. The step-wise standard operating procedure for each is described below:
The Front System
- To start the procedure, sign in the log book with the necessary information including name and time.
- Check the temperature of the cooling water circulator maintained at 25°C.
- The laser key should be turned to the on position from the standby position.
- The wavelength must be changed by adjusting the dial button and the select button is pushed.
- The machines and the computer are turned on and the software is run.
- The PMT high voltage and Pockels cell controller in the software are set to 0.
- The sample is placed on the stage and checked with a bright field and epifluorescence.
- The room light is turned off and the filter cube wheel is turned to position 1 and the path selection bar is pulled out.
- The interlock system is turned on by closing the sliding front panel.
- The laser shutter is opened and the live scan is opted for. The PMT voltage is increased and the image window shows a non-zero value.
- The cell controller is gradually increased and the image shows up. The PMT power, the cell controller, duration, and image preference can be adjusted as per the manual.
- Details of power value, cell voltage, and laser wavelength are checked and noted in the log book.
- To close down the system, the filter cube wheel is positioned to 2-6, and the path selection bar is pushed in.
- The PMT voltage and the Pockels controller are set to 0.
- The program is turned off and the laser shutter is closed. Lastly, the laser key is turned to the standby position.
The Back System
- The first four steps of the standard startup procedure for the front system are the same for the back system.
- Then, the computer and the hardware are turned on. The multiphoton microscopy is run.
- The PMT high voltage is set to off while the laser intensity is set to 0.7.
- The sample specimen is placed on the stage and the room light is turned off. The filter cube is positioned to position 1 and the light path selector knob is pulled out.
- The laser shutter is opened from the laser controller in the hardware and PMT is checked.
- Opt to continuous scan and the OMT high voltage is increased to 70-80% and the image window shows a non-zero value.
- The laser intensity is gradually increased from 0.7 from the software. The PMT power, laser intensity, duration, and image preference can be adjusted as per the manual.
- To close down the system, the PMT high voltage is set to off and the laser intensity is set to zero from the software. The filter cube wheel is positioned to 2-6, and the path selector knob is pushed in.
- The program and the laser shutter are closed. Lastly, the laser key is turned to the standby position.
Applications of Multiphoton Microscopy
- Multiphoton microscope is widely used in functional biology research, especially for imaging cell interactions in transgenic animals. It is used to study biological structures, rates of cell trafficking, and localization of fluorescent cells.
- It has been used to visualize the bone, bone marrow, and vital anatomical structures through second harmonic generation.
- Certain metal and metal oxide nanoparticles are particularly compatible with multiphoton microscopy. This is due to their enhanced photoluminescence in multiphoton settings and can assist in distinguishing nanoparticle signals from autofluorescence signals.
- Similarly, the integration of fluorescence lifetime imaging with multiphoton microscopy has been increasingly used in dermatology applications. It has been utilized in the observation of healthy skin, aging skin, and in the diagnosis of basal cell carcinoma.
- As it is used to image in vivo, it is beneficial in diagnosing various pathological conditions in neuroscience, immunology, gastroenterology, and oncology.
- It is widely used to observe the movements and interactions of living T- and B-cells, along with the antigen-presenting cells, in a living organism.
Examples of Multiphoton Microscope
1. Ultima 2Pplus
- It offers high adaptability, resolution, imaging depth, and speed, enabling users to efficiently and effectively conduct simultaneous imaging, stimulation, and electrophysiology experiments.
- This system is specifically engineered for intravital imaging, featuring fully motorized control for the X-Y-Z positioning of the objective and two rotational axes for accurate imaging alignment.
2. FEMTO3D Atlas
- It is a combination of advanced science and engineering that is used to achieve 3D measurements.
- It allows users to examine 3D structures of cells and various biological processes, such as neuronal and dendritic activities at speeds up to a million times faster than conventional scanning techniques and maintains two-photon resolution.
3. Bergamo® III Series
- It is a modular system arranged for two- and three-photon imaging.
- This device is used in vitro and in vivo, encompassing deep brain imaging, spatial light modulation, electrophysiology, expansive field of view, rapid imaging, etc.
4. Ultima Investigator Plus
- It is based on a technique designed for live imaging enhancement.
- It features an increased field-of-view and an improved detection pathway.
- It integrates three-photon imaging, multi-region scanning, and motorized nosepieces providing a thorough imaging experience to the users.
Advantages of Multiphoton Microscopy
- This technique utilizes longer wavelengths for excitation, like red or infrared, which typically generates a larger volume of illumination.
- It provides non-invasive image contrast.
- An increase in depth penetration, reduction in photobleaching, phototoxicity, and sample damage contribute significantly to the overall quality of imaging in biological studies.
- Underlying cells and living tissues can be observed for a longer duration.
- It has higher fluorescence collection efficiency and greater signal strength at any tissue depth as both excitation and emission occur exclusively at the focal plane.
- Multiphoton microscopy allows the observation of lymphoid organs in six-dimensional imaging including X, Y, Z, time, intensity, and wavelength.
Limitations of Multiphoton Microscopy
- The multiphoton microscopy system is too bulky and expensive due to its equipment.
- Although known for its advantage in reducing photobleaching and photodamage, these problems may arise due to mechanical breakdown.
- The cell damage can increase with a decrease in pulse width and an increase in average power.
Precautions
- Professional training for the user is required to operate multiphoton microscopy.
- Avoid staring at the laser beam without adequate safety as the IR laser is not visible to the naked eye.
- Avoid exposing skin to the path of the laser beam.
- It is mandatory to turn off the room light and avoid exposing it to PMT high voltage.
- The water circulator cooling must be turned on before turning the laser on.
Conclusion
Multiphoton microscopy has become a powerful tool with advancements in cell-based biological studies including molecular cell biology, genomics, and proteomics. Its capability to visualize deep within intact cells and organs is one of the many benefits compared to other microscopy techniques. Its applications extend beyond fundamental research and clinical medicine, including pathological diseases and metabolic disorders diagnosis through the examination of tissue at a cellular resolution.
With the significant developments in introducing new fluorophores, more targeted probes, and laser technologies, future improvements in multiphoton microscopy will be a milestone in unlocking numerous opportunities in the field of research and life science. In addition, its integration with other imaging techniques has been considered to be beneficial in biomedical research, and physiology. In conclusion, multiphoton microscopy is a remarkable breakthrough in imaging technology, potentially enhancing our understanding of cellular interactions and disease mechanisms.
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