We have demonstrated a straightforward and noninvasive method to identify the distribution of grana
and starch within an intact leaf. Grana and starch are the major functional structures for
photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular
structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana
and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence
(2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and
starch while 2PF from chlorophyll indicates the identity of grana.
Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed in vivo to trace the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. Furthermore, using video-rate THG microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-cells, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imaging may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status.
The understanding of the interaction between tumors and surrounding microenvironment in vivo is an important first step
and basis for pathway-targeting cancer therapy. To in vivo observe the dynamic development of tumor cells and validate
the efficacy of therapy in microscopic scales, people commonly performed multi-photon fluorescence microscopy
through an invasive window chamber setup. However, under such system, the cancer cells can't be identified and
long-term tracked without a fluorescence labeling. Exploiting the intrinsic third harmonic generation (THG) and
two-photon fluorescence (2PF) contrasts of melanin, we demonstrated in vivo identification of melanoma and tracked its
development without labeling. It was achieved with a least invasive femtosecond Cr:forsterite laser and a laser scanning
nonlinear microscopy system with 3D sub-micron spatial resolution. Combined with molecular probes or reporters, we
anticipate thus developed platform a powerful tool to reveal molecular insights of tumor microenvironments, enhance
our understanding of tumor biology, and trigger new therapeutic approaches.