Stain-free histopathology by programmable supercontinuum pulses

Abstract

The preparation, staining, visualization, and interpretation of histological images of tissue is well-accepted as the gold standard process for the diagnosis of disease. These methods were developed historically, and are used ubiquitously in pathology, despite being highly time and labor intensive. Here we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic crystal fiber source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate collection of optical signatures of the tumor microenvironment, including evidence of mesoscopic biological organization, tumor cell migration, and (lymph-)angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use.

Raman spectroscopy: techniques and applications in the life sciences

“…SRS is also compatible with other nonlinear optical microscopy techniques such as CARS, second/third harmonic generation and multiphoton fluorescence which share similar instrumentation and usually require simple changes in the detection configuration for measurement. This was demonstrated recently using a programmable supercontinuum laser source for multi-modal nonlinear microscopy, of which SRS was one component, for ex-vivo imaging of cancerous mammary tissue from rats [259]. Drug delivery has been monitored several times with SRS microscopy, where the often unique and strong Raman features of the drugs can easily be separated from the biological Raman bands using multi-color SRS.…”

Spectrally-broad coherent anti-Stokes Raman scattering hyper-microscopy utilizing a Stokes supercontinuum pumped at 800 nm

“…CARS occurs when at least two light pulses-pump/probe and Stokes, having respective frequencies of ω P,Pr and ω S -interact in a non-linear material such that the difference frequency, Ω R = ω P,Pr -ω S , matches a vibrational mode of the material, resulting in the resonant generation of photons at the anti-Stokes frequency ω AS = 2ω P,Pr -ω S [1][2][3]. With its combination of rapid imaging with rich spectroscopic information, CARS "hyper-microscopy" is gaining popularity in research fields such as biomedical imaging [4][5][6], materials science [7], and biology [8][9][10][11].…”

“…Such frequency-scanning CARS can thus be thought of as "imaging first", where rapid-even video rate [13]-image collection enables live-cell imaging, albeit at the expense of sparse or time-consuming hyperspectral capabilities. A hybrid technique that utilizes both single-wavelength detection and broadband laser excitation is known as spectralfocusing CARS (SF-CARS) [5,[14][15][16]. With SF-CARS, a narrowband pump pulse and broadband Stokes pulse are stretched in time such that their frequency-time relationship (i.e.…”

“…the probed CARS frequency-can be simply scanned by sweeping the relative temporal overlap between the two pulses. Because it utilizes single-wavelength detection, SF-CARS is predominantly an imaging-first technique; and since the spectrum can be scanned in a matter of seconds-tominutes [5,10,16,17], it can yield good-quality hyperspectral image stacks at a rate that challenges multiplex-CARS techniques.…”

“…SF-CARS has been implemented using three distinct experimental approaches: by using two synchronized laser sources to generate pump/probe and Stokes [15]; by using a single spectrally-broad sub-25-fs laser source with intra-pulse filtering to generate the pump/probe and Stokes [16,18]; or by using a fs-laser-pumped photonic crystal fibre (PCF) source to generate a supercontinuum from which some (or all) of the pump/probe/Stokes beams are derived [5,9,10,14,19]. Implementations of SF-CARS that use a single spectrally-broad femtosecond laser source have been successful in performing both CARS imaging and dense spectral acquisition, albeit with the added challenges and costs of utilizing an ultra-broadband laser system.…”