© 2025 Journal of Forensic Science and Medicine | Published by Wolters Kluwer ‑ Medknow 26 Original Article INTRODUCTION The determination of the sequence of crossing ink lines means to determine the order in which two ink lines intersect each other while writing on a paper. It is an essential part of questioned document examination when the document is in question of being altered without the authentication of the owner. [1] Throughout the years, researchers have segregated various aspects of it to study the morphologic analysis of the ink lines. There is insufficient knowledge concerning the physicochemical analysis of two writing inks intersecting on a sheet of paper. A number of influencing factors contribute to the complexity of this aspect, including type and color of ink and paper, type of writing instrument, and variety of cross‑combinations. Speed, pressure, dragging of ink, direction or shape of stroke, or the time between writing also plays a critical role in stroke formation. [2] There are different techniques for the visualization of the sequence of strokes. The most commonly used one is spectroscopy. The order of line crossing has been successfully determined using the combined technologies of focused ion beam and scanning electron microscope/energy dispersive X‑ray instrument by some researchers. Various spectroscopic and nondestructive techniques such as absorption/reflectance spectra, docubox dragon, electrostatic detection apparatus, stereomicroscope, video spectral comparator, infrared spectroscopy, atomic force microscopy, field emission scanning electron microscope with energy‑dispersive X‑ray spectroscopy, and Raman spectroscopy have been utilized for the sequence examination till date. There are mainly two types of intersections, i.e., homogenous and heterogeneous. A homogeneous line intersection is the result from writing with two similar inks, and a heterogeneous line intersection is the result of using two different writing inks. Fluorescence spectroscopy is a promising analytical method for analysis of ink since it combines light microscopy’s magnifying capabilities with the ability to see the fluorescence. A fluorescence microscope, in contrast to a regular microscope, uses a significantly A Forensic Approach in the Determination of Sequence of Strokes using Fluorescence Spectrometry Vartika Khare 1 , Anu Singla 1 , Pawan Gupta 2 1 Dr. APJ Abdul Kalam Institute of Forensic Science and Criminology, Bundelkhand University, Jhansi, Uttar Pradesh, India, 2 Uttar Pradesh State Institute of Forensic Science, Lucknow, Uttar Pradesh, India Background: Fluorescence Microscope is a promising analytical method for the examination of the sequence of strokes of writing inks of different colours, brands, and models in a questioned document. Aims and Objectives: In the present article, an attempt has been made to categorize the writing inks of different brands and colours on the basis of their fluorescence. Materials and Methods: A total of 36 writing inks were used to make the different intersections on a standard A4 sheet which were then analyzed under the excitation filters of the Fluorescence Microscope. Results: The results indicated that the different inks can be characterized optically based on the luminescent components of the ink present in crossed lined intersections. Conclusions: Fluorescence Microscopy is a relatively simple, fast, inexpensive, and non‑destructive technology that can be used in conjunction with other, more complex, and expensive technologies to identify the sequence of line crossing. It can provide a novel way of analysis based on the assessment of microelement analysis and the characteristics of diverse ink handwriting samples. Keywords: Fluorescence microscope, forensic science, handwriting, ink discrimination, questioned documents, sequence of strokes Access this article online Quick Response Code: Website: https://journals.lww.com/jfsm DOI: 10.4103/jfsm.jfsm_43_23 Address for correspondence: Miss. Vartika Khare, Dr. APJ Abdul Kalam Institute of Forensic Science and Criminology, Bundelkhand University, Kanpur Road, Jhansi ‑ 284 128, Uttar Pradesh, India. E‑mail: vartikakhare22@gmail.com This is an open access journal, and arcles are distributed under the terms of the Creave Commons Aribuon‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creaons are licensed under the idencal terms. For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com How to cite this article: Khare V, Singla A, Gupta P. A forensic approach in the determination of sequence of strokes using fluorescence spectrometry. J Forensic Sci Med 2025;11:26‑31. Abstract Submitted: 09‑Apr‑2023 Revised: 07‑Jul‑2023 Accepted: 02‑Aug‑2023 Published: 28‑Feb‑2025