A revolution in biological imaging that harnesses natural fluorescence to observe living cells without disruptive dyes.
For centuries, scientists peering through microscopes have faced a fundamental dilemma: how to see the intricate details of living cells without altering or destroying them in the process. Traditional methods often required adding fluorescent dyes—foreign substances that could disrupt delicate biological processes.
What if we could instead harness the natural glow of molecules already present within cells? This dream is now reality through a revolutionary technology that uses ultrafast lasers to illuminate life's fundamental building blocks in their natural state.
Welcome to the fascinating world of multiphoton microscopy, where scientists are using incredibly brief laser pulses to unlock the hidden glow within biological substances, revealing nature's secrets without disturbing its delicate balance.
Many biological substances naturally emit light when properly excited, a phenomenon known as endogenous fluorescence. Unlike methods that require adding foreign dyes, endogenous fluorescence harnesses the natural glow of molecules already present in cells and tissues 1 .
Key biological substances like the amino acid tryptophan, elastin, collagen, and keratin possess this innate ability to fluoresce. Tryptophan, fundamental to protein synthesis, is particularly important as it serves as a built-in marker for studying biological structures and processes without external labels 1 .
Multiphoton microscopy represents a quantum leap in imaging technology. In conventional fluorescence, a single high-energy photon excites a molecule. In multiphoton excitation, a molecule simultaneously absorbs two or three longer-wavelength (lower-energy) photons, combining their energy to reach the excited state 9 .
This process offers significant advantages including deeper tissue penetration, reduced background noise, and minimal cell damage, enabling longer observation of living processes.
The fluorescence process occurs in three stages, beautifully illustrated by what scientists call a Jablonski diagram 7 . First, a molecule absorbs energy from light, elevating it to an excited state. Next, it undergoes subtle changes during its brief excited-state lifetime (typically 1-10 nanoseconds). Finally, it returns to its ground state by emitting a photon of lower energy—this is the fluorescence we detect 7 .
A picosecond laser emits optical pulses with durations between 1 and tens of picoseconds (a picosecond is one trillionth of a second) 2 . These ultrafast lasers create the extremely high peak powers necessary for multiphoton processes while maintaining low average power that living tissues can tolerate 9 .
The most advanced systems use mode-locked solid-state lasers or fiber lasers to generate these brief pulses 2 . For three-photon microscopy particularly, researchers often turn to ytterbium-based fiber laser systems that can produce pulses with energies 40-50 times higher than conventional two-photon lasers 9 .
A groundbreaking experiment demonstrated the power of this technology by imaging the endogenous fluorescence of tryptophan using both two- and three-photon excitation 1 . The research team developed an original solution specifically dedicated to multiphoton microscopy, centered around an ultrawide band laser system with a unique filtering system based on a prism-line that allowed spatial shaping of the spectrum 1 .
The experimental setup involved several sophisticated components:
This custom-designed system, coupled with a picosecond ultrawide band laser correctly filtered spectrally, was specifically adapted for characterizing the two- and three-photon absorption ranges and imaging of tryptophan 1 .
The experiment yielded compelling results that highlighted the advantages of three-photon imaging. Researchers successfully demonstrated that using a picosecond ultrawide band laser spectrally filtered does allow reaching both the two- and three-photon absorption ranges of tryptophan 1 .
Most strikingly, a quantitative comparison of the resulting images showed significant differences in image quality. The three-photon images appeared better contrasted and better resolved than the two-photon ones 1 . This notable improvement in image quality can be explained by studying the probability of presence of multiphoton processes involved and the cross-section values of three-photon absorption versus two-photon absorption 1 .
| Laser Parameter | 2-Photon | 3-Photon |
|---|---|---|
| Wavelength Range | 700-1100 nm | 1300-1700 nm |
| Average Power | 1-2 W | 1-4 W |
| Pulse Width | 75-150 fs | 40-60 fs |
| Pulse Energy | 10-50 nJ | 1-2 μJ |
| Repetition Rate | 50-100 MHz | 1-4 MHz |
This initiating work, cumulating an innovative multiphoton setup and interesting results, plays a crucial role for extending the label-free imaging ability of multiphoton microscopy 1 . The successful demonstration of three-photon excitation of endogenous fluorophores like tryptophan opens new possibilities for non-invasive biological imaging.
The implications of advanced multiphoton microscopy extend far beyond basic research. The ability to perform label-free imaging of endogenous fluorophores opens new possibilities across multiple fields:
In neuroscience, three-photon microscopy can image deeper into the brain, potentially revolutionizing our understanding of neural circuits 9 .
In medical diagnostics, researchers are exploring fluorescence spectroscopy for analyzing erythrocytes, potentially leading to non-invasive blood analysis methods .
Recent breakthroughs have demonstrated that fluorescent proteins can be turned into quantum bits (qubits) within cells, potentially enabling a new generation of biological quantum sensors 4 .
Looking ahead, scientists are already exploring even more sophisticated applications. Though this technology currently requires cryogenic temperatures, it hints at a future where quantum physics and biology merge to give us unprecedented views of cellular processes.
The development of multiphoton microscopy with picosecond ultrawide band lasers represents more than just a technical achievement—it offers a new philosophy for exploring biological systems. By harnessing endogenous fluorescence, scientists can now observe life's processes with minimal interference, watching the intricate dance of molecules in their native environment.
As laser technologies continue to advance and our understanding of biological fluorescence deepens, we stand at the threshold of even more remarkable discoveries. The inner glow of life's building blocks, once hidden from view, is now illuminating a path toward a deeper understanding of the complex machinery that drives living systems.
| Tool/Reagent | Function |
|---|---|
| Ultrafast Lasers | Generate high-intensity pulses for multiphoton excitation |
| Optical Parametric Amplifiers (OPA) | Convert laser wavelengths to optimal ranges for 3-photon imaging |
| Fluorescence Detectors | Capture emitted photons from samples |
| Endogenous Fluorophores | Natural contrast agents for label-free imaging |
| Spectral Filters | Separate emission light from excitation light |