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Pathology with Third Harmonic Generation Microscopy


Gap between Pre-operative imaging and pathology

Cytological& histological

features observed with SHG/THG imaging

THG imaging:

Third-harmonic generation microscopy

We use Third Harmonic Generation microscopy to create instant sharp 3D images of tissue.


Nonlinear microscopy without fluorescent labels

Laser-induced nonlinear process provides contrast localized to the laser focus, since excitation scales with I²-³. 3D-imaging by scanning the focus through the sample. The different signals, THG, SHG and 2- or 3-photon excited autofluorescence are separated by spectral filtering.


arrow left Normal brain tissue

Cells and cell nuclei can be seen clearly.

arrow left Transition zone in low-grade glioma tissue

3D images up to a depth of several 100’s of micrometers can be obtained.

THG/SHG image of low-grade glioma tissue arrow right

Large areas can be scanned, of up to 10 x 10 mm², to detect the boundaries of a glioma by looking at the variation in cell density [2].

H&E stained image of same area low-grade glioma brain tumor tissue arrow right 

THG microscopy images show a very good agreement with the golden standard in pathology, H&E analysis where cell density is an indicator of the state of the tissue [2].

arrow left SHG/THG image of a blood vessel

With THGM we can scan through blood vessels, observe the blood vessel wall, muscle layer, elastin layer, and blood cells.

arrow left SHG/2PF image of skin tissue, side view

In skin tissue, connective tissue together with hair and hair follicle are visualised (depth stack of 100 µm).

Stacked image of fat tissue. arrow right

In fat tissue, fat cells are clearly visible, surrounded by connective tissue

Muscle tissue of chicken arrow right

THG/SHG imaging on the tip of a needle is in development for in-situ imaging [2].


[1] N.V. Kuzmin, S. Idema, E. Aronica, P.C. de Witt Hamer, P. Wesseling, M.L. Groot, Higher harmonic generation imaging for neuropathology, in: HANDBOOK OF NEUROPHOTONICS, F.S. Pavone and S. Shoham, eds., Taylor & Francis Books, Inc. Accepted for publication.

[2] N.V. Kuzmin, P. Wesseling, P.C. de Witt Hamer, D.P. Noske, G.D. Galgano, H.D. Mansvelder, J.C. Baayen, M.L. Groot, Third harmonic generation imaging for fast, label-free pathology of human brain tumors, Biomed Opt Express 7 (2016), 1889-1904.

[3] S. Witte, A. Negrean, J.C. Lodder, C.P. de Kock, G. Testa Silva, H.D. Mansvelder, M.L. Groot, Label-free live brain imaging and targeted patching with third-harmonic generation microscopy, Proc Nath Acad Sci USA 108 (2011), 5970-5975.

[4] Z. Zhang, N.V. Kuzmin, M.L. Groot, and J.C. de Munck, Extracting morphologies from third harmonic generation images of structurally normal human brain tissue, Bioinformatics 33 (2017), 1712-1720.

[5] Z. Zhang, N.V. Kuzmin, M.L. Groot, and J.C. de Munck, Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images, J Biophotonics (2017).

THG as clinical tool

“Recognize tumor boundaries intraoperatively”

Fast, label-free, no tissue preparation ✔
Detection in backscatter direction ✔
Mosaic imaging: from um to mm scale ✔
THG images show histopathological information ✔

In association with

We are currently working with the most specialized companies in the Netherlands.
Innovation Exchange Amsterdam

Tritos Diagnostics B.V.

W&N Building, Room T-275
De Boelelaan 1081
1081 HV Amsterdam
The Netherlands

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