Advanced Imaging Microscopy • Spectroscopy • Ultrafast optics
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Research blog of Juan David Muñoz-Bolaños

Advancing imaging through optical physics & computation

Microscopes, applications and theory of Raman, two-photon, coherent Raman and photothermal imaging, adaptive optics, and the open-source tools SpMap and 2D Optics.

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Microscopes
6 modalities
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Applications
6 fields
📐
Theory
5 lecture notes
📝
Blog
8 articles
💻
Software
SpMap · 2D Optics
Adaptive optics confocal Raman setup
Featured research · Dec 2024

Confocal Raman Microscopy with Adaptive Optics

First application of adaptive optics in confocal Raman microscopy: a transmissive optofluidic wavefront shaper on the nosepiece corrects sample-induced aberrations, boosting Raman signal up to 3.5x in mouse brain tissue. (ACS Photonics)

Muñoz-Bolaños J.D., Rajaeipour P., Kummer K., Kress M., Ataman Ç., Ritsch-Marte M., Jesacher A. ACS Photonics, doi:10.1021/acsphotonics.4c01432

Read the full article →

Latest articles

All 8 articles →
1064 nm Fiber Probe Raman Imaging for human bladder resectates
Clinical Raman•Jul 7, 2024

1064 nm Fiber Probe Raman Imaging for human bladder resectates

A compact dispersive 1064 nm fiber-probe Raman imaging spectrometer that suppresses autofluorescence, discriminates tumor from non-tumor bladder tissue and measures kidney stones. (Applied Sciences)

Read full article →
Optofluidic spatial light modulator in multi-photon imaging
Multiphoton•Feb 10, 2023

Optofluidic spatial light modulator in multi-photon imaging

Optofluidic adaptive optics in multiphoton microscopy: a transmissive, low-loss phase modulator for aberration correction in two-photon imaging. (Biomedical Optics Express)

Read full article →
SpMap: Hyperspectral package for spectroscopists
Software•Aug 15, 2022

SpMap: Hyperspectral package for spectroscopists

Open-source Python 3 package for hyperspectral images: preprocessing, baseline correction, clustering and spectral unmixing for Raman, IR and fluorescence data.

Read full article →

Open-source software

Details →
SpMap
Open source · PyPI Python 3

SpMap

Hyperspectral analysis for spectroscopists

Load, preprocess and analyse Raman, IR and fluorescence hyperspectral images: smoothing, baseline correction, normalisation, PCA, k-means and hierarchical clustering, and spectral unmixing. Used for the bladder-cancer Raman study.

pip install spectramap
View on PyPI Documentation post
2D Optics 3D ray trace of a Raman spectrometer
Open source · GitHub Python · Qt

2D Optics

Optical design & ultrafast optics workbench

Desktop application for designing microscopes and spectrometers: sequential 2D/3D ray tracing with a lens data editor, spot diagrams, ray fans and RMS wavefront; damped-least-squares and global optimisation; gratings and DMDs; ultrafast pulse analysis (spectral phase, GDD, pulse duration); and a dual-pulse nonlinear imaging workbench for SFG, DFG, CARS and SRS. Exports STEP/DXF CAD.

◉ Ray tracing & optimisation ◉ Ultrafast pulse analysis ◉ CARS / SRS / SFG simulation ◉ Spectrometer & DMD design
git clone https://github.com/MicroscopeMaestro/2D_Optics.git
View on GitHub

Imaging Microscopes

The optical microscopes and spectroscopic imaging modalities we build and use, from spontaneous and coherent Raman to two-photon and photothermal microscopy, with adaptive optics throughout.

Confocal Raman Microscopy
λ

Confocal Raman Microscopy

Label-free chemical imaging with diffraction-limited 3D sectioning. Our system adds sensorless adaptive optics via an optofluidic phase plate on the objective nosepiece.

Spontaneous RamanConfocal sectioningAdaptive optics
  • 📄 Raman Imaging
  • 📝 Confocal Raman Microscopy with Adaptive Optics
  • 📝 Raman Intensity Calibration from 500 to 1100 nm laser excitation
  • 📝 Raman Wavenumber Calibration by paracetamol
1064 nm Fiber-Probe Raman Imaging
∿

1064 nm Fiber-Probe Raman Imaging

Dispersive NIR Raman imaging through a fiber probe. 1064 nm excitation suppresses tissue autofluorescence for clinical specimens.

1064 nm excitationFiber probeClinical ex vivo
  • 📄 Raman Imaging
  • 📝 1064 nm Fiber Probe Raman Imaging for human bladder resectates
Two-Photon Excitation Fluorescence (2PEF)
2γ

Two-Photon Excitation Fluorescence (2PEF)

Femtosecond-laser scanning microscopy with intrinsic optical sectioning and deep penetration, combined with holographic and adaptive wavefront control.

fs NIR laserLaser scanningWavefront shaping
  • 📄 Two-Photon Excitation Fluorescence (2PEF)
  • 📝 TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)
  • 📝 Optofluidic spatial light modulator in multi-photon imaging
Coherent Raman (CARS / SRS)
ω

Coherent Raman (CARS / SRS)

Pump and Stokes pulses drive molecular vibrations coherently for video-rate chemical imaging. Designed and simulated with the 2D Optics nonlinear-imaging workbench.

Dual-pulseUltrafastVideo-rate chemistry
  • 📄 Advanced Raman Imaging (CARS / SRS)
ΔT
ΔT

Photothermal Microscopy

A pump beam is absorbed and heats the sample locally; a probe beam reads the resulting refractive-index change, imaging non-fluorescent absorbers.

Pump-probeAbsorption contrastLabel-free
  • 📄 Photothermal Microscopy
Adaptive Optics & Wavefront Shaping
≈

Adaptive Optics & Wavefront Shaping

Optofluidic SLMs, deformable phase plates and holographic methods (DASH, PMC) that restore focus quality deep inside scattering samples.

Optofluidic SLMSensorless AOHolography
  • 📄 Two-Photon Excitation Fluorescence (2PEF)
  • 📝 Confocal Raman Microscopy with Adaptive Optics
  • 📝 Optofluidic spatial light modulator in multi-photon imaging
  • 📝 TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)

Applications

Where these microscopes make a difference: clinical diagnostics, neuroscience, deep-tissue imaging, metrology and hyperspectral data science.

Cancer diagnostics

Cancer diagnostics

Raman imaging discriminates tumor, necrosis and healthy tissue in human bladder resectates without labels, supporting surgical decisions.

  • 📝 1064 nm Fiber Probe Raman Imaging for human bladder resectates
Neuroscience & brain tissue

Neuroscience & brain tissue

Adaptive optics corrects aberrations in mouse brain tissue, recovering resolution and up to 3.5x Raman signal.

  • 📝 Confocal Raman Microscopy with Adaptive Optics
Deep-tissue in vivo imaging

Deep-tissue in vivo imaging

Holographic wavefront shaping and optofluidic AO extend two-photon imaging depth and speed in scattering tissue.

  • 📄 Two-Photon Excitation Fluorescence (2PEF)
  • 📝 TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)
  • 📝 Optofluidic spatial light modulator in multi-photon imaging
Urology: kidney stones

Urology: kidney stones

1064 nm excitation acquires clean spectra of highly fluorescent kidney stones that 785 nm systems cannot measure.

  • 📝 1064 nm Fiber Probe Raman Imaging for human bladder resectates
Spectral metrology & calibration

Spectral metrology & calibration

Wavenumber and intensity calibration protocols so spectra are reproducible across instruments, lasers and laboratories.

  • 📝 Raman Wavenumber Calibration by paracetamol
  • 📝 Raman Intensity Calibration from 500 to 1100 nm laser excitation
Hyperspectral data science

Hyperspectral data science

Clustering, unmixing and machine learning turn hyperspectral cubes into chemical maps, implemented in SpMap.

  • 📄 Machine Learning for Hyperspectral Images
  • 📝 SpMap: Hyperspectral package for spectroscopists
  • 📝 PDF LaTeX equations to LaTeX code by deep learning (pix2tex)

Theory

Lecture-style notes on the physics behind each technique, plus methods articles on calibration, wavefront shaping and machine learning.

Theory · Lecture notes

Raman Imaging

The Raman effect, vibrational spectroscopy and how Raman maps are acquired and processed into chemical images.

Read →
Theory · Lecture notes

Advanced Raman Imaging (CARS / SRS)

Coherent Raman scattering: CARS and SRS, why they are orders of magnitude faster than spontaneous Raman, and their instrumentation.

Read →
Theory · Lecture notes

Two-Photon Excitation Fluorescence (2PEF)

Nonlinear excitation, intrinsic optical sectioning, deeper penetration and reduced phototoxicity in two-photon microscopy.

Read →
Theory · Lecture notes

Photothermal Microscopy

Imaging absorption through the local heating it produces: label-free contrast for non-fluorescent materials and biological samples.

Read →
Theory · Lecture notes

Machine Learning for Hyperspectral Images

Supervised and unsupervised methods (PCA, clustering, unmixing, classifiers) for Raman, IR and fluorescence hyperspectral data.

Read →

Methods & tutorials

TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)
Wavefront Shaping•Jan 16, 2022

TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)

Scattering limits in vivo depth. DASH and PMC holographic wavefront-shaping methods recover a focus deep in scattering tissue for two-photon excitation fluorescence.

Read full article →
Raman Intensity Calibration from 500 to 1100 nm laser excitation
Calibration•Mar 5, 2022

Raman Intensity Calibration from 500 to 1100 nm laser excitation

How to calibrate the intensity (y) axis of Raman spectra with calibration lamps and reference standards so results are reproducible across instruments and excitation wavelengths.

Read full article →
Raman Wavenumber Calibration by paracetamol
Calibration•Jan 12, 2022

Raman Wavenumber Calibration by paracetamol

Wavenumber (x-axis) calibration at 785 nm using paracetamol reference peaks, with polynomial pixel-to-wavenumber fitting.

Read full article →
PDF LaTeX equations to LaTeX code by deep learning (pix2tex)
Machine Learning•Jan 17, 2022

PDF LaTeX equations to LaTeX code by deep learning (pix2tex)

Convert printed (PDF) equations into editable LaTeX code instantly with the pix2tex vision-transformer model.

Read full article →

Blog

All 8 published articles, newest first. Click any article to read it in full with all figures.

Confocal Raman Microscopy with Adaptive Optics
Adaptive Optics•Dec 20, 2024

Confocal Raman Microscopy with Adaptive Optics

First application of adaptive optics in confocal Raman microscopy: a transmissive optofluidic wavefront shaper on the nosepiece corrects sample-induced aberrations, boosting Raman signal up to 3.5x in mouse brain tissue. (ACS Photonics)

Read full article →
1064 nm Fiber Probe Raman Imaging for human bladder resectates
Clinical Raman•Jul 7, 2024

1064 nm Fiber Probe Raman Imaging for human bladder resectates

A compact dispersive 1064 nm fiber-probe Raman imaging spectrometer that suppresses autofluorescence, discriminates tumor from non-tumor bladder tissue and measures kidney stones. (Applied Sciences)

Read full article →
Optofluidic spatial light modulator in multi-photon imaging
Multiphoton•Feb 10, 2023

Optofluidic spatial light modulator in multi-photon imaging

Optofluidic adaptive optics in multiphoton microscopy: a transmissive, low-loss phase modulator for aberration correction in two-photon imaging. (Biomedical Optics Express)

Read full article →
SpMap: Hyperspectral package for spectroscopists
Software•Aug 15, 2022

SpMap: Hyperspectral package for spectroscopists

Open-source Python 3 package for hyperspectral images: preprocessing, baseline correction, clustering and spectral unmixing for Raman, IR and fluorescence data.

Read full article →
Raman Intensity Calibration from 500 to 1100 nm laser excitation
Calibration•Mar 5, 2022

Raman Intensity Calibration from 500 to 1100 nm laser excitation

How to calibrate the intensity (y) axis of Raman spectra with calibration lamps and reference standards so results are reproducible across instruments and excitation wavelengths.

Read full article →
PDF LaTeX equations to LaTeX code by deep learning (pix2tex)
Machine Learning•Jan 17, 2022

PDF LaTeX equations to LaTeX code by deep learning (pix2tex)

Convert printed (PDF) equations into editable LaTeX code instantly with the pix2tex vision-transformer model.

Read full article →
TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)
Wavefront Shaping•Jan 16, 2022

TPEF: Improving speed and deep tissue imaging by holography (DASH and PMC)

Scattering limits in vivo depth. DASH and PMC holographic wavefront-shaping methods recover a focus deep in scattering tissue for two-photon excitation fluorescence.

Read full article →
Raman Wavenumber Calibration by paracetamol
Calibration•Jan 12, 2022

Raman Wavenumber Calibration by paracetamol

Wavenumber (x-axis) calibration at 785 nm using paracetamol reference peaks, with polynomial pixel-to-wavenumber fitting.

Read full article →

Open-Source Software

Tools we develop and share: SpMap for hyperspectral data analysis and 2D Optics for optical, ultrafast and nonlinear microscope design.

SpMap
Open source · PyPI Python 3

SpMap

Hyperspectral analysis for spectroscopists

Load, preprocess and analyse Raman, IR and fluorescence hyperspectral images: smoothing, baseline correction, normalisation, PCA, k-means and hierarchical clustering, and spectral unmixing. Used for the bladder-cancer Raman study.

pip install spectramap
View on PyPI Documentation post
2D Optics 3D ray trace of a Raman spectrometer
Open source · GitHub Python · Qt

2D Optics

Optical design & ultrafast optics workbench

Desktop application for designing microscopes and spectrometers: sequential 2D/3D ray tracing with a lens data editor, spot diagrams, ray fans and RMS wavefront; damped-least-squares and global optimisation; gratings and DMDs; ultrafast pulse analysis (spectral phase, GDD, pulse duration); and a dual-pulse nonlinear imaging workbench for SFG, DFG, CARS and SRS. Exports STEP/DXF CAD.

◉ Ray tracing & optimisation ◉ Ultrafast pulse analysis ◉ CARS / SRS / SFG simulation ◉ Spectrometer & DMD design
git clone https://github.com/MicroscopeMaestro/2D_Optics.git
View on GitHub
2D Optics grating diffraction trace

2D Optics in practice

Traced first-order diffraction from a 600 lines/mm grating in a NIR Raman spectrometer designed in 2D Optics. Each wavelength is followed through the lenses, grating and DMD to the detector to check focus, vignetting and dispersion before anything is built.

The same environment models femtosecond pulses (dispersion and pulse width along every ray path) and simulates dual-pulse CARS/SRS microscopes from calibrated component data.

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Author
Juan David Muñoz-Bolaños
Biophotonics & Advanced Microscopy
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Advanced Imaging
Juan David Muñoz-Bolaños · Leibniz Institute of Photonic Technology
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